Deoxyribonucleic acid (DNA) is the self-replicating hereditary material that provides a blueprint which, in collaboration with environmental influences, produces a structural and functional phenotype. As DNA coordinates and directs differentiation, growth, survival, and reproduction, it is responsible for life and the continuation of our species. Genome integrity requires the maintenance of DNA stability for the correct preservation of genetic information. This is facilitated by accurate DNA replication and precise DNA repair. DNA damage may arise from a wide range of both endogenous and exogenous sources but may be repaired through highly specific mechanisms. The most common mechanisms include mismatch, base excision, nucleotide excision, and double-strand DNA (dsDNA) break repair. Concurrent with regulation of the cell cycle, these mechanisms are precisely executed to ensure full restoration of damaged DNA. Failure or inaccuracy in DNA repair contributes to genome instability and loss of genetic information which may lead to mutations resulting in disease or loss of life. A detailed understanding of the mechanisms of DNA damage and its repair provides insight into disease pathogeneses and may facilitate diagnosis and the development of targeted therapies. Deoxyribonucleic acid (DNA) is the hereditary material found in humans, other eukaryotes, and prokaryotes that carries instructions for structure and function . Acting The purpose of the cell cycle is to generate two genetically identical daughter cells from a single parent cell . This is1) phase, the DNA synthesis (S) phase, and the pre-mitotic/gap 2 (G2) phase, while mitosis comprises the mitotic (M) phase ..1 phase,1 checkpoint ensures that cell size is adequate, that nutrient supply is sufficient, that growth factors are present, and that there is no DNA damage [2 checkpoint ensures that error-free DNA replication occurs by activating DNA repair mechanisms during an induced pause in the cycle if required [The GA damage ,18. The required ,18. Nearrequired ,18.DNA damage is defined as chemical (dynamic) and physical  alterations to the DNA double helix that are derived from endogenous or exogenous origins and impair the function and integrity of DNA ,20.If the damaged DNA is repairable, the necessary cell cycle checkpoints are activated, the DNA is repaired, genome integrity is restored, and the cell survives . If the N-glycosidic bonds between nitrogenous bases and deoxyribose residues, resulting in apurinic and apyrimidinic site formation [Endogenous DNA damage, originating from internal metabolic processes, includes damage caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) ,20. Thesormation ,20,23. Iormation ,20. Replormation . The intormation . Furtherormation ,27.Exogenous DNA damage, originating from external environmental processes, includes ionizing and solar ultraviolet radiation ,20. IoniThe DDR is an integrated signaling and genomic maintenance network which enables cells to withstand threats posed by DNA damage ,29,30. T1, S, and G2 phases of the cell cycle, thus mediating DNA repair and cell death [The DNA damage checkpoint complex is composed of sensors, signal transducers, and effector pathways . The funll death ,32.367, Ser1893, and Ser1981 [3016 [ATM is an important protein involved in the activation of cell cycle checkpoints ,35. ATM  Ser1981 . In addi81 [3016 . As a re81 [3016 ,39. Cell81 [3016 ,11.1/S phase arrest [2/M phase arrest [2/M phase transition [The ATM protein is activated by dsDNA breaks, whereas the ATR protein responds to single-strand DNA (ssDNA) breaks ,40. ATM e arrest . Chk2 ace arrest ,42. Inhie arrest . Chk1 alansition ,42. ATM,ansition . p53 phoansition ,32. Cellansition ,32.Cell cycle checkpoint prolongation and DDR protein recruitment is highly dependent on the characteristics and complexity of the DNA damage sustained ,43. TherMismatch repair is responsible for correcting base pair mismatches which occur when adenosine-guanine and cytosine-thymidine do not pair correctly . The speIn initiating mismatch repair, the Mutator S (MutS) complex is responsible for detecting DNA mismatches ,49,50. TIn the recognition step, MutS is recruited ahead of proliferating cell nuclear antigen (PCNA), an essential DNA replication accessory protein . MutSα (2−) and hydrogen peroxide (H2O2)), X-rays , alkylating agents , and spontaneous reactions  [Base-excision repair is involved in the removal and replacement of damaged DNA bases . Tomas Ltalling) ,57. DNA talling) ,58.2) during aerobic respiration [2O2 is a by-product of numerous biochemical reactions such as uric acid formation, but may also be generated by ionizing radiation [2O2 produces two oxidized base products, namely 8-oxoguanine, which binds to adenine or cytosine to form a transversion mutation (conversion from a purine to a pyrimidine and vice versa), and thymine glycol which inhibits DNA replication [ROS are generated during normal cellular respiration ,59. Elecpiration . ROS havpiration . H2O2 isadiation . H2O2 prlication ,59.N-glycosidic bond between deoxyribose and the damaged base [In base-excision repair, a substrate-specific DNA glycosylase enzyme detects a damaged DNA base and removes it by cleaving the ged base ,60,61,62ged base ,60,61,62ged base ,60,61,62ged base ,60,61,62ged base ,60,61,62ged base ,60,61,621 phase continuing throughout the S phase [1 phase and declines in the S phase [Base excision repair corrects large numbers of small DNA base lesions caused by alkylation, deamination, and oxidation reactions ,44. Comp S phase ,58. The  S phase ,58.N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine), which are generated from exogenous carcinogen metabolism and endogenous lipid peroxidation, as well as intrastrand crosslinks, characterised by the covalent binding of nucleotides within the same DNA strand  from DNA by excising an oligonucleotide containing the lesion to replace it with newly synthesised DNA . The disA strand  [43,65,6DNA double helix-distorting lesions are recognized by the xeroderma pigmentosum group A (XPA) protein and undergo repair in one of two pathways depending on the type of lesion i.e., transcription-coupled nucleotide-excision repair or global genome nucleotide excision repair ,67. DNA Global genome nucleotide excision repair is initiated by the xeroderma pigmentosum group C-RAD23 homolog B (XPC-RAD23B) complex which binds to the non-damaged DNA strand opposite to the lesion ,69,70. TDsDNA breaks occur when the sugar-phosphate backbones of both DNA strands are broken at a similar position or in close proximity to one other . Subsequ139 resulting in the formation of foci at sites of DNA damage lending to the recruitment of repair proteins [The MRN complex detects dsDNA breaks and subsequently recruits and activates members of the DDR machinery such as those of the phosphatidylinositol 3 kinase (PI3K) family ,75. Actiproteins ,77. Althproteins .With dsDNA breaks, two principle mechanisms may be implemented in the repair process, namely NHEJ and HR . This clCS) [CS-interacting protein Artemis endonuclease trimming DNA ends or polymerases filling DNA ends  ,82. NHEJCS) ,82,83. DDNA ends  [81,82,8A paralog of XRCC4 and XLF (PAXX), a member of the XRCC4 superfamily, is recruited to dsDNA damage breaks and interacts directly with the Ku70/Ku80 heterodimer to initiate NHEJ ,86. More2 phases of the cell cycle when replicated sister chromatids are available [BRCA1) and 2 (BRCA2) [In HR, replicated sister chromatid DNA sequences are used as templates to restore missing DNA sequences on the damaged chromatid ,88. For vailable ,88. HR ivailable ,88,89. R (BRCA2) ,87,88,89 (BRCA2) . RAD51 c (BRCA2) . Broken  (BRCA2) . As HR m (BRCA2) .Two additional dsDNA break repair pathways exist that require sequence homology, namely single-strand annealing (SSA) and alternative non-homologous end-joining  . SSA is Aurora kinase A and ninein-interacting protein (AUNIP) acts as a dsDNA damage sensor and interacts with CtIP to ensure adequate accumulation of CtIP at dsDNA breaks to initiate DNA end resection and subsequently HR ,98. AUNIUbiquitin-specific protease 4 (USP4) facilitates HR by directly participating in dsDNA break end-resection through the post-translational process of autoubiquitination ,100. USPRare hereditary diseases characterized by DNA repair deficiencies arise when repair machinery and mechanisms are defective . GermlinMismatch repair deficiencies are present in adult-onset autosomal dominant Lynch syndrome and child-onset autosomal recessive constitutional mismatch repair deficiency syndrome, which include well-established colorectal and endometrial cancer syndromes ,101,102.The identification of various pathologies related to inadequate DNA damage detection and DNA repair mechanisms highlights the fundamental role these processes play in maintaining genomic stability . These nA defective DDR system is a hallmark of certain cancers which allows tumour cells to proliferate and acquire mutations . These D+) as a substrate to synthesize poly (ADP-ribose) chains by an autoregulated process [PARP1 binds to damaged DNA ends via two homologous N-terminal zinc (Zn) finger domains, Zn1 and Zn2 . The car process . A struc process . Poly  as a monotherapy for women diagnosed with regimens . The admregimens . Olapariregimens ,126. Olaregimens .DNA strand break bait (Dbait) molecules are DNA repair inhibitors that mimic dsDNA breaks and sequester dsDNA break repair proteins such as DNA-PK and PARP1 . These lBiau et al. (2014) conducted a preclinical study in which a cholesterol-conjugated Dbait molecule, DT01, sensitized melanoma cells to radiotherapy both in vitro and in vivo . In addiPARP inhibitors constitute a major emerging class of promising therapeutics; however, various other DNA repair pathway inhibitors are also currently being investigated . PreclinGenetic engineering is an emergent experimental treatment strategy with potential applications in incurable genetic disorders . One areAlthough the potential clinical applications of CRISPR/Cas9 are numerous, these are in early stages of research . CRISPR/DNA is responsible for carrying hereditary information across generations; it accomplishes this by controlling the production and function of proteins. As a result, it is essential for growth, survival, and reproduction. Should aberrations occur in DNA, genome integrity is maintained through accurate DNA replication and adequate DNA repair. DNA damaging agents may be of endogenous or exogenous origin and the resulting DNA lesions may cause morbidity and mortality if not repaired. Specific DNA repair mechanisms that are closely associated with the cell cycle exist to correct the different types of DNA lesion that occur. Failure of these vital repair processes may lead to a variety of mutations and, consequently, diseases. Through an understanding of the causes of DNA damage and the corresponding repair mechanisms, it is possible to design strategies to create or improve methods for the prevention, diagnosis, and treatment of pathologies related to deficiencies in the mechanisms of DNA repair.


Montelukast inhibited cell proliferation and colony formation and induced the cell death of lung cancer cells. Further investigation showed the down-regulation of B-cell lymphoma 2 (Bcl-2), up-regulation of Bcl-2 homologous antagonist/killer (Bak), and nuclear translocation of apoptosis-inducing factor (AIF) in montelukast-treated lung cancer cells. Montelukast also markedly decreased the phosphorylation of several proteins, such as with no lysine 1 (WNK1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (Erk1/2), MAPK/Erk kinase (MEK), and proline-rich Akt substrate of 40-kDa (PRAS40), which might contribute to cell death. In conclusion, montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous cohort study. The utility of montelukast in cancer prevention and treatment thus deserves further studies.Developing novel chemo-prevention techniques and advancing treatment are key elements to beating lung cancer, the most common cause of cancer mortality worldwide. Our previous cohort study showed that cysteinyl leukotriene receptor antagonists, mainly montelukast, decreased the lung cancer risk in asthma patients. In the current study, we conducted in vivo and in vitro experiments to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. Using Lewis lung carcinoma-bearing mice, we showed that feeding montelukast significantly delayed the tumor growth in mice ( Cancer is a leading cause of morbidity and mortality ,2,3. AltIn our previous nationwide population-based cohort study, we found that cysteinyl leukotriene receptor antagonists (LTRAs) decreased cancer risk in a dose-dependent manner in asthma patients, and the significant chemo-preventing effect of LTRA was mainly observed in lung, breast, colorectal, and liver cancers . LTRAs, As dysregulated cell death and increased proliferation are associated with carcinogenesis, one of the most important approaches to develop novel anti-cancer treatment is discovering therapeutic agents that induce cancer cell death. Historically, cell death was classified into apoptosis and necrosis . CaspaseThe major aim of this study was to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. To further confirm the cytotoxic effect of montelukast on lung cancer cells, we treated a variety of lung cancer cell lines with various concentrations of montelukast. Using a Water-Soluble Tetrazolium Salt-1 (WST-1) Cell Proliferation Assay, we found that 100 μM of montelukast induced more than 75% of growth inhibition in A549, H1299, H460, CL1-0, CL1-5, and Lewis lung carcinoma (LLC) a,b. FurtTo investigate the possible mechanisms of the montelukast-induced cell death of lung cancer cells, the expression levels of apoptosis-associated proteins were analyzed with immunoblot. Montelukast treatment markedly decreased the expression of Bcl-2 and markedly increased the expression of Bak in a time-dependent manner in A549 and CL1-5 a,b. HoweTo investigate whether apoptosis-inducing factor (AIF) participates in montelukast-induced cell death, its levels in the nuclei were assessed. Montelukast markedly increased the levels of AIF in the nuclear fragments a–c. UsinWe screened the protein phosphorylation pattern with a Human Phospho-Kinase Array Kit to investigate the possible regulator of montelukast-induced cell death and found a markedly decreased phosphorylation of WNK1, Akt, Erk1/2, MEK, and PRAS40 in montelukast-treated CL1-5 a. Using p < 0.0001)  a. The tu 0.0001) b,c and i 0.0001) d,e than In our previous study analyzing the Taiwan National Health Insurance Database, we demonstrated that LTRAs, mainly montelukast, decreased cancer risk in a dose-dependent manner in asthma patients, and the cancer risk reduction was mainly observed in lung, breast, colorectal, and liver cancers . The fin4 pathway has been recognized in many cancers, such as prostatic cancer [4, LTD4, and LTE4, which are originally recognized for their effects in promoting bronchoconstriction, inflammation, mucus secretion, and microvascular permeability, have also been gradually understood for their contribution in carcinogenesis and cancer progression [4, mainly through the stimulation of its principle receptor, cysteinyl leukotriene receptor 1 (CysLT1R), promotes cell survival, proliferation, migration, and the transcription of potentially oncogenic genes [4 level than healthy subjects [4 treatment augmented the growth of the xenograft tumor  in nude mice [Cysltr1) also demonstrated the important role of CysLT1R in colon carcinogenesis [1R was noted in specimens from gastric cancer [1R was correlated to poor prognosis in patients of breast cancer [Inflammatory immune responses are crucial mediating mechanisms in the tumor microenvironment . The croc cancer , esophagc cancer , head anc cancer , gastricc cancer , and colc cancer . Cysteingression ,20. LTD4ic genes . Patientsubjects . LTD4 trude mice . A recenogenesis . Clinicac cancer , renal cc cancer ,8, transc cancer ,9, prostc cancer ,10, and c cancer ,11. Furtt cancer  and colot cancer ,37. 1R in various cancer cells, including renal cell carcinoma [1 phase and apoptosis [1 phase and apoptosis of the cancer cells [Some in vitro studies have demonstrated the overexpression of CysLTarcinoma ,8, transarcinoma ,9, prostarcinoma ,10, testarcinoma ,11, and arcinoma , while mpoptosis . Anotherer cells . In linen-nitroso n-methyl urea-inducing mammary carcinogenesis also demonstrated the chemo-preventive effect of montelukast [Only a few in vivo studies in the literature to date have shown the chemo-preventive effect of leukotriene pathway inhibitors ,13,14,15telukast . In lineThe mechanisms through which montelukast induces cancer cell death have not yet been well studied. As shown in a study using human neuroblastoma cells, montelukast induced the cleavage of caspase 9 and caspase 3 . The stu4-induced activation of MEK1/2 and Erk1/2 in cell proliferation [Many signaling pathways involved in cell survival and proliferation are usually dysregulated in cancer cells. Mitogen-activated protein kinase (MAPK) cascades, especially those involving extracellular signal-regulated kinase (Erk) 1/2 activated by MAPK/Erk kinase (MEK) 1/2 dual-specificity protein kinases, promote cancer cell survival and migration ,43. In lferation ,45, the feration , the monferation ,47,48,494 might induce the up-regulated expression and activation of COX-2 via the activation of Erk1/2 [Most previous studies showed that LTDf Erk1/2 ,44. Intef Erk1/2 . This mif Erk1/2 ,50. Besif Erk1/2 . TherefoThe dosing of current anti-cancer drugs is usually limited by the potential side effects, leading to less treatment effects. How to make the most of the current anti-cancer drugs remains an important issue. Combining few drugs in lower doses, adding adjuvant agents, and selecting a responsive population are common strategies. For example, our previous study showed that C-X-C motif chemokine 5 (CXCL5) antibody synergistically enhanced the effect, without increasing the toxicity, of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in treating lung cancer . Given iHuman lung cancer cells, including A549, H460, and H1299, were obtained from the American Type Culture Collection . Less invasive (CL1-0) and highly invasive (CL1-5) human lung adenocarcinoma cell lines were generously provided by Prof. Pan-Chyr Yang . A549 was cultured in modified Eagle’s medium (MEM)  supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin . H460, H1299, CL1-0, and CL1-5 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin . Mouse Lewis lung carcinoma cell line, LLC1 (LL/2), was obtained from ATCC and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) , which was supplemented with 10% FBS and penicillin/streptomycin (100 U/0.1 mg/mL) . Montelukast was purchased from Sigma-Aldrich Co. . Reagents were dissolved in dimethyl sulfoxide (DMSO) . Control cultures received the carrier solvent (0.1% DMSO), unless otherwise specified. Antibodies were purchased from Cell Signaling Technology, Inc.  and Millipore Co. . All other chemicals used were of the purest form available commercially.Using a Premixed WST-1 Cell Proliferation Assay , cell proliferation/viability was determined. In brief, the cells were incubated with the reagent for 0.5–4 h. In viable and metabolically active cells, the tetrazolium salt WST-1 was cleaved to a formazan-class dye by mitochondrial succinate-tetrazolium reductase . TherefoTo determine the long-term effect of montelukast, 1000 cells were seeded in a 6-cm dish and incubated overnight to attach. Cells were treated with montelukast or vehicle control for 24 h and then incubated in fresh medium for nine days (medium was replaced every three to four days). Colonies were stained with crystal violet  and the number of colonies was counted.The cells were treated with montelukast or vehicle control. Followed by two times of washing with ice-cooled PBS, the cells were then fixed in 4% paraformaldehyde for 10 min. After being stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and washed with PBS for two times, they were examined under Leica DM IL LED with a DC100 Digital Camera . For each slide, at least two representative low-power fields were captured (both light microscopy and fluorescence microscopy) and the percentages of cells with shrinking nuclei were calculated.g for 15 min, the supernatant fraction was collected for immunoblot. Equivalent amounts of protein were resolved by SDS-PAGE (8–12%) and transferred to polyvinylidene difluoride membranes. After blocking for 1 h in 5% non-fat dry milk in tris-buffered saline, the membrane was incubated with the desired primary antibody for 1–16 h. After being treated with the appropriate peroxidase-conjugated secondary antibody, the immune-reactive proteins were detected using an enhanced chemiluminescence kit  according to the manufacturer’s instructions. Each membrane was also divided or stripped to examine the level of loading control (GAPDH or lamin A/C).The cells were lysed on ice for 60 min by RIPA lysis buffer  . After the cell lysate was centrifuged at 14,000× https://imagej.nih.gov/ij/), the gels were analyzed with densitometry. The average values of multiple replicates, which were normalized to the corresponding internal controls, are presented along with the representative figures.Using ImageJ  according to the manufacturers’ instructions.® 488 goat anti-rabbit antibody  (1:200) in the dark at room temperature for 1 h, followed by incubation with DAPI (1:1000) in the dark at room temperature for 5 min. Slides were sealed with coverslips and then examined under a Zeiss LSM 700 confocal microscope .To demonstrate the nuclear translocation of apoptosis-inducing factor (AIF), 70,000 cells were seeded in each well of the Millicell EZ Slides  and incubated overnight to attach. Cells were treated with montelukast or vehicle control for 24 h, followed by two times of washing with ice-cooled phosphate-buffered saline (PBS). The cells were then fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 for 15 min, and then blocked in PBS supplemented with 5% bovine serum albumin (BSA) overnight. After washing twice with 1% BSA in PBS (1% BSA-PBS), the cells were probed for AIF with anti-AIF antibody (1:100) overnight at 4 °C. After washing with 1% BSA-PBS, slides were incubated with Alexa FluorThe cells were treated with 0.1% DMSO (control) or 60 μM montelukast for 8 h. The levels of protein phosphorylation in the cell lysates were assessed with a Human Phospho-Kinase Array Kit  according to the manufacturers’ instructions.The C57BL/6JNarl (B6) (four to six weeks old) were purchased from the National Laboratory Animal Center  and maintained in a specific pathogen-free condition in the Experimental Animal Center under the supervision of board-certified veterinarians. After adapting for a week in the Experimental Animal Center, the mice were used for experiments. All animal experiments followed the protocol approved by the Institutional Animal Care and Use Committee (IACUC) .The mice were given either water (control group) or 100 μg of montelukast in water (treatment group) via tube feeding daily from day −3 to day 13. Each mouse was injected with 500,000 LLC cells subcutaneously on day 0. In order to determine the tumor volume by an external caliper, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined; the subcutaneous tumor volume based on caliper measurements was calculated by the modified ellipsoidal formula (tumor volume = 0.5 × (length × width × width)) . To invep-values less than 0.05 were considered significant. The extraction and computation of data, data linkage, processing and sampling, and statistical analyses were performed using SAS system . The statistical significance level was set at a two-sided p value of <0.05.Data were expressed as mean values with standard deviations (means ± SD) unless otherwise indicated. Statistical comparisons of the results were made using analysis of variance (ANOVA). All comparisons were two-tailed, and Montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous study , as well


Cronobacter sakazakii serogroup O:2, sequence type 64. Assemblies for these strains have sizes of 4,442,307 and 4,599,266 bp and % G+C contents of 56.9 and 56.7, respectively.We introduce draft genome sequences of strains CDC1121-73  and GK1025 (powdered infant formula manufacturing facility isolate), which are both malonate-positive  Cronobacter genus comprises seven species, three of which  have been implicated in cases of severe infantile septicemia and meningitis infections, as well as wound and urinary tract infections in adults (C. sakazakii (Csak) strains that are associated with foods was under appreciated, possibly leading to misidentification of strains when relying on phenotypic identification alone . The CDC1121-73 and GK1025 WGS assemblies were found to have 70 and 94 contigs (>500 bp long), 56.9 and 56.7% G+C content, and 4,112 and 4,295 CDS, respectively. WGS assemblies for CDC1121-73 and GK1025 were annotated using the RAST annotation server  was performed on the isolates using the MiSeq platform , using Illumina’s NextSeq XT library kit. Trimmed Fastq data sets were cmeB, acrR, mcaA, and arsenic resistance were found. Each strain had 11 alleles related to stress response, for example, degS and universal stress protein genes A-C, E, and G and 24 alleles for polysaccharide biosynthesis, transportation, and exportation. PCR results support genomic findings which show that both strains possessed the pESA3-like virulence plasmid and additionally, strain CDC1121-73 harbored a pCTU3-like plasmid  as Biosamples SAMN04329632 and SAMN04329637, SUBIDs 1743736 and 1743738, and assemblies MCOD00000000 and MCOE00000000, respectively.The accession numbers of


CYCLIN D expression, CDK4/6 activation, and the repression of cyclin-dependent kinase inhibitor proteins (CDKi) by EGFR signaling pathways. We also discuss the successes and challenges of EGFR-targeted therapies, and the potential for their use in combination with CDK4/6 inhibitors.The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly upregulated in cancers such as in non-small-cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer. Various mechanisms mediate the upregulation of EGFR activity, including common mutations and truncations to its extracellular domain, such as in the EGFRvIII truncations, as well as to its kinase domain, such as the L858R and T790M mutations, or the exon 19 truncation. These EGFR aberrations over-activate downstream pro-oncogenic signaling pathways, including the RAS-RAF-MEK-ERK MAPK and AKT-PI3K-mTOR pathways. These pathways then activate many biological outputs that are beneficial to cancer cell proliferation, including their chronic initiation and progression through the cell cycle. Here, we review the molecular mechanisms that regulate EGFR signal transduction, including the EGFR structure and its mutations, ligand binding and EGFR dimerization, as well as the signaling pathways that lead to G1 cell cycle progression. We focus on the induction of  Epidermal growth factor (EGF) receptor (EGFR), also known as ErbB1/HER1, is the prototype of the EGFR family that also includes ErbB2/HER2/Neu, ErbB3/HER3, and ErbB4/HER4 [Stanley Cohen’s Nobel Prize-winning studies provided the seminal work for the EGFR field. In 1959, together with Rita Levi-Montalcini, Cohen was examining the effects of a newly discovered growth factor, the nerve growth factor (NGF), on newborn mice. Their source of NGF consisted of crude extracts prepared from mouse salivary glands. Dr. Levi-Montalcini had noticed that unexpected “side effects” were present in whole fraction preparations unrelated to the effects seen in NGF-purified fractions . The resThe EGFR’s link to cancer was first recognized when the transforming v-ErbB oncogene of the avian erythroblatosis virus was found to be a mutant homolog of human EGFR ,11. The It is well established that the ErbB family of proteins can promote a multitude of pro-oncogenic biological processes, including cell proliferation, angiogenesis, inhibition of apoptosis, cell motility, adhesion, and metastasis. It is clear that ErbB family members represent promising targets for pharmacological inhibition. Various cancer treatment options targeting mutated or overexpressed ErbBs have reached the clinics, and tend to show efficacy and improve long term outcome. However, it has become evident that cancer cells can employ various molecular mechanisms to escape cell death mediated by anti-ErbB treatments. The characterization of these pathways has taken centre stage, in hoping that these discoveries will lead to effective combination therapies.The human ErbB gene family includes EGFR/ERBB1/HER1, NEU/ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4. Being able to form homo- and heterodimers with one another, these family members can form a total of 28 different combinations with each other . ERBB2 dCaenorhabditis elegans, the ErbB gene is a single receptor, with a single ligand [Drosophila, ErbB is made up of one receptor and four ligands [Drosophila have yielded tremendous information, especially regarding embryogenesis [In e ligand . In Dros ligands . EGFR paogenesis .6 receptors per cell is observed in cancer cells [EGFR is mapped to chromosome 7 short arm q22, spanning 110 kb of DNA divided into 28 exons ,32. In ner cells .EGFR promoter include E1A [EGFR gene expression [EGF regulates its own receptor, as it increases EGFR RNA expression  by stimulude E1A , Sp1 [36lude E1A , and AP2lude E1A . The intpression .Almost all cell types possess ErbB family members, with the exception of hematopoietic cells . EGFR faIn adolescence, EGFR family genes play key roles in mammary ductal development. Female mice with a T743G substitution that impairs tyrosine kinase activity fail to develop proper mammary glands, due to defective ductal growth, causing their pups to die from malnutrition ,58,59.EGFR activity remains high in most parts of mature CNS . HoweverThe EGFR is synthesized as a 1210 residue precursor that is cleaved at the N-terminal to result in the mature 1186 residue transmembrane EGFR . Here, wThe extracellular region of the EGFR is composed of 621 amino acids and is subdivided into four domains, I , II , III , IV . Domains I and III are leucine-rich fragments that participate in ligand binding. Domain II forms homo- or hetero-dimers with the analogous domain of family members. Domain IV can form disulfide bonds to domain II, and links to the TM domain. Domains II and IV do not make contacts with the ligand, and are cysteine-rich regions.The TM domain is a 23 amino acid long hydrophobic single pass membrane structure, that anchors the receptor to the membrane . It is 2The intracellular domain is 542 amino acids long, and includes the flexible juxtamembrane segment (~40 aa), the tyrosine kinase domain , and the C-terminal tail  . The tyrEGFR mutations occur at mutational “hotspots” in the extracellular region, the kinase domain, and the C-terminal tail . CertainEGFR ectodomain oncogenic alterations often cause the loss of the inhibitory regulatory domains for dimerization. The most famous EGFR ectodomain mutant is the viral EGFR homologue, v-ERBB, which completely lacks the ectodomain, and exists primarily in dimers . In addiM for ATP than wild type EGFR [EGFR exon 19 in-frame deletions, commonly observed in NSCLC [In the kinase domain, the most commonly seen EGFR point mutation is L858R, and makes up approximately 45% of mutations in the tyrosine kinase domain ,82. Termype EGFR ,84. Moleype EGFR ,85. Anotin NSCLC . Anotherin NSCLC ,88. Whilin NSCLC . T790M iin NSCLC .The first studies on the EGF in animals showed that it stimulated epidermal proliferation and keratinization . We now Human EGF is a 6 kDa protein made up of 53 amino acids. Physiologically in humans, various organs regulate their innate EGF concentrations . For exaAside from the EGF, six other EGFR ligands have been described. These include transforming growth factor-α (TGF-α), amphiregulin (AREG), epiregulin (EREG), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), and epigen (EPI). EGF, TGF-α, and amphiregulin are specific ligands only for the EGFR . TGF-α iEach ligand appears to activate the EGFR in the same way: through ligand binding, receptor dimerization, receptor transautophosphorylation, and the recruitment of signaling proteins or adaptors. In addition, all EGFR ligands induce EGFR internalization and trafficking to early endosomes . DespiteMost growth factors act in a paracrine fashion, where the growth factor produced from a cell targets a neighboring cell. However, examples of autocrine mechanisms have been documented. Originally elaborated by Sporn and Roberts , this daFor full EGFR activation, ligand binding and EGFR dimerization are crucial. However, it was a matter of controversy which process occurred first. It is now well accepted that for the activation of the EGFR, EGF-binding to each EGFR monomer precedes EGFR dimerization. Originally observed by Yarden and Schlessinger ,113, thec domain or of a dimeric leucine zipper. The characterization of the interactions between EGFR domains remains difficult since many experiments are carried out predominantly with isolated domains, either extracellular, TM, or intracellular. As such, it is difficult to understand the synergy between each domain throughout ligand activation [There is evidence that preformed unliganded EGFR dimers do exist ,117,118,tivation .The site of EGFR dimerization is considered “receptor-mediated”, in that the ligand does not make a direct contribution to the dimerization interface . Each EGAlthough EGFR has been proclaimed as a prototypical, its unique “receptor-mediated” mechanism of dimerization and its ability to transautophosphorylate make it anything than such. These subtle differences lead to approaches by which pharmaceutical targeting of the EGFR could take advantage of these facts.The signal transduction pathways activated by the EGFR comprise the most important reasons the EGFR has been studied to such lengths. The powerful capacity of each of the multitude of pathways under the EGFR’s control to drive cell proliferation and resist apoptosis has formed a strong motivation for their cancer-related researches. Large strides have been accomplished in elucidating the pathways involved in mediating EGFR activity. Current research largely focuses on the crosstalk and compensation between pathways, so to elucidate how cancer cells develop resistance to pharmacological inhibition of pathways and how to properly intervene. The metabolic alterations mediated by these pathways are also of focus. Here, we will review the activation mechanisms by which the EGFR activates the ERK MAPK, AKT-PI3K, and PLC-γ1-PKC pathways, focusing on the molecular interactions between each protein in the pathways . We willOf the four family members, the EGFR signals to the largest number of unique signaling pathways, including the ERK MAPK, PI3K-AKT, SRC, PLC-γ1-PKC, JNK, and JAK-STAT pathways. As these pathways are inter-linked, the activation of the EGFR actually stimulates an entire signaling network associated with a wide number of outcomes, such as cell proliferation, growth, differentiation, migration, and inhibition of apoptosis. Many proteins within the EGFR’s signal transduction network have been the subject of pharmaceutical targeting in malignancies, illustrating the potency of the receptor.Binding of EGF to the EGFR leads to the transphosphorylation of various tyrosine residues on the intracellular C-terminal tail. The tyrosine residues phosphorylated by EGF addition to cells include Y703, Y920, Y992, Y1045, Y1068, Y1086, Y1148, and Y1173. In addition to these autophosphorylated sites, there are also residues that are phosphorylated by other kinases, which interestingly appear downstream in the EGFR-activation cascade. For example, Y845 is phosphorylated by c-SRC , and T65The RAS-RAF-MEK-ERK MAPK pathway may be the most important pathway in mediating the biological response of the EGFR. Bonafide proto-oncogenes RAS and RAF reside in this pathway. A major target of cancer therapy is MEK. ERK MAPK (mitogen-activated protein kinase) interacts with over a hundred substrates to initiate a wide array of physiological and pathological responses, including growth, proliferation, differentiation, migration, and inhibition of apoptosis ,127. SubOverview of RAS-ERK MAPK PathwayFollowing receptor transphosphorylation, the activated EGFR’s Y1068 and Y1086 residues bind directly to GRB2 (growth factor receptor binding protein 2), by its SH2 domain from residues 60–158 ,129,130.SHC and GRB22, it is more efficiently recruited by PA. GRB2 can also bind CBL and CIN85, two important proteins for EGFR trafficking [Receptor transphosphorylation forms binding sites for both SHC and GRB2 adaptor proteins. Complexes that contain SHC have been reported to be better at activating RAS than those without SHC  . Recruitfficking ,155,156.fficking . Althougfficking .SOS and RAS2, and PA. In addition, non-substrate RAS can form a binding site for SOS recruitment to the PM, which can potentiate the important GTP exchange activity of SOS for RAS by 500-fold [As mentioned, SOS recruitment to the PM requires GRB2, PIP500-fold . Moreove500-fold . The exc500-fold . It was 500-fold . On the 500-fold . Therefo500-fold . RAS actRAS genes [Active RAS has the ability to activate three major downstream effectors. These include RAF-1 , PI3K, and RalGDS (Ras-like guanine nucleotide-dissociation stimulator) ,165,166.AS genes . HoweverAS genes . While tAS genes ,169,170.RAFRAS itself does not fully activate RAF. Rather, RAS initiates RAF activation by translocating it to the plasma membrane, where additional membrane-localized activation events occur. RAS recruits RAF through the RBD region. In addition to this site, the RAS cysteine-rich domain  has been shown to be important for the efficient activation of RAF-1 ,173,174.EGF treatment recruits 14-3-3-associated RAF-1 to the PM and leads to dissociation of 14-3-3 from RAF-1 . It has MEK and ERKMEK1 and MEK2 are activated by RAF-1, its only known substrates . FurtherCYCLIN D1 promoter to activate transcription of the important G1 driver [ERK1 and ERK2 are serine/threonine kinases that always appear to be activated together . Unlike 1 driver ,217 .The PI3K-AKT-mTOR signaling cascade controls metabolism, proliferation, cell size, survival and motility. In cancer, this pathway is often hyper-activated due to activating mutations to EGFR family members, PI3K, AKT, and downregulation of the famous tumour suppressor PTEN, which antagonizes PI3K activity. The PI3K-AKT-mTOR pathway is also dysregulated in diabetes, autism, and aging .2) to generate phosphatidylinositol-3,4,5-triphosphate (PIP3) [3 is a potent secondary messenger and is the predominant mediator of PI3K activity [3 is highlighted by the fact that the PI3K antagonist PTEN, which dephosphorylates and limits the activity PIP3. is frequently inactivated in cancer [3 links the lipid kinase activity of PI3K to the network of downstream signaling pathways, including the PH (pleckstrin homology) domain-containing serine/threonine kinase AKT/PKB. PI3K also indirectly stimulates the production of phosphatidylinositol-3,4-bisphosphate P2), which can also recruit PH domain-containing proteins, including AKT. Other than AKT, cells contain 50–100 downstream effectors of PI3K [PI3K was discovered in the 1980s by the Cantley group . It was e (PIP3) ,228,229.activity . The strn cancer . PIP3 li of PI3K . Other n of PI3K .The recruitment of PI3K differs between ErbB family members following ligand stimulation. PI3K binds directly with ERBB3 and ERBB4 ,234, but3. AKT binds to PIP3 through its PH domain [AKT  is a serine/threonine kinase with a wide variety of substrates that impact cell survival, proliferation, metabolism, protein synthesis, growth, and migration. It is activated by a dual regulatory mechanism, requiring its translocation to the PM and phosphorylation at two conserved residues. Activated EGFR stimulates AKT translocation to the PM by activating the PI3K-induced formation of PIPH domain . In addiH domain . Once loH domain ,246. PDKH domain . Active H domain . PhosphoH domain ,249. AKTH domain ,251, andH domain .Pten haplodeficient mice [AKT2 is prominently observed [There are three members of the AKT family: AKT1, AKT2, and AKT3. AKT2 and AKT3 have 81% and 83% amino acid homology to AKT1 respectively . AKT isoent mice . On the ent mice ,272,273.observed ,274,275.Regarding the regulation of AKT isoforms under the EGFR, all isoforms appear to be under the control of PI3K upon EGF stimulation in various esophageal cancer cells . HoweverAKT mediates its wide range of physiological responses through the activation or deactivation of several downstream proteins. Here, we discuss its role in mediating cell survival, in activating mTOR, and in metabolism. The role of AKT in cell proliferation, including its inhibition of GSK-3β (glycogen synthase kinase-3 beta), is discussed in L protein [In terms of cell survival, AKT functions in an anti-apoptotic manner by directly phosphorylating components of the cell death machinery. AKT phosphorylates the pro-apoptotic BAD (Bcl-2-associated death promoter) protein at S136, which inactivates BAD and prevents it from binding and inhibiting the anti-apoptotic BCL-X protein . AKT als protein , as well protein . FOXO1 d protein . AKT als protein ,283,284.One of the most important AKT pathways is to signal to mTOR . mTOR refers to two distinct complexes: mTORC1 is made up of raptor, GbL/mLST8, and negative regulatory subunits PRAS40 and DEPTOR, whereas mTORC2 contains rictor, mSin1, and Protor, GbL/mLST8 and DEPTOR . mTORC1 In the past decade, more research has been done to understand the role of the PI3K-AKT-mTOR pathway in the control of cell metabolism and glycolysis in cancer. In normal cells, high oxygen levels favor the use of oxidative phosphorylation over glycolysis, as it is a more efficient mechanism for generating ATP. Tumour cells however continuously use glycolysis even at high oxygen levels, a term coined aerobic glycolysis and also the principle behind the phenomenon known as the Warburg effect . Althoug2 focused on the results of its receptor-mediated hydrolysis by phospholipase C (PLC) [Plcg1 null knockout mouse is embryonic lethal at E9.0, in part due to the impairment of vasculogenesis [Plcg1, has been shown to upregulate cell migration and invasion in vitro and in vivo, including upregulating metastasis in cancer [Prior to the discovery of PI3K, interest on PIP C (PLC) . Thirtee C (PLC) . Notablyogenesis ,304. PLCn cancer ,306.3 formed by PI3K in response to EGF stimulation [2 into free intracellular 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), two important secondary messengers. IP3 binds to IP3-receptors at the endoplasmic reticulum to induce intracellular calcium release. Calcium release converges with the DAG pathway, as both DAG and Ca2+ activate protein kinase C (PKC). Phosphorylation at the activation loop of the kinase domain of PKC by PDK1 is necessary for its activation, and the specific residues that are phosphorylated have been characterized [PLC-γ1 binds directly to activated EGFR at Y992 and Y1173 using its SH2 domain ,308,309.mulation ,311. Phomulation ,313. Onccterized . For exacterized ,316. PKCcterized . Interescterized . Anothercterized ,319,320.cterized ,322 Phoscterized . PA can cterized ).The first virus ever identified to cause cancer encoded a viral isoform of c-SRC . The isoform, discovered in 1911 by Francis Peyton Rous, was named v-SRC, as it induced sarcomas in chickens . v-SRC lc-SRC is widely implicated in various aspects of EGFR signaling. For example, it can directly phosphorylate EGFR Y845, RAF Y341, SHC1, clathrin, and CBL . It is nSince SRC and EGFR appear to cooperate to increase tumorigenicity, dual inhibition of SRC and EGFR has been proposed, such as in head and neck squamous cell carcinoma and colorectal cancer ,353. How2O2, UV, therapeutic agents, or ionizing radiation cause the EGFR to translocate to the nucleus, with nuclear EGFR signaling playing roles in cell proliferation, tumor progression, DNA repair [Several lines of evidence have shown that stimulation with EGF, as well as HA repair ,359,360.A repair ,362,363,A repair ,365,366.Nuclear EGFR appears to be the full-length receptor . The mecCYCLIN D1 and c-MYC, among other proto-oncogenes [CYCLIN D1 gene promoter has been better studied, and has been shown to require EGFR interaction with two proteins, Mucin-1 (MUC-1) and RNA helicase A (RHA) [In the nucleus, the EGFR supports gene regulation by acting as a transcriptional co-activator. it has been shown that nuclear EGFR aids in the transcription of important cell cycle progression mediators, including ncogenes ,370. The A (RHA) ,372. In The EGFR and its signaling pathways play key roles in oncogenesis. Two major classes of EGFR-targeted therapies have been developed. The first class of therapy developed includes humanized monoclonal antibodies against the EGFR extracellular domain, designed to block ligand binding or to mediate its downregulation . The secAnti-EGFR therapy generally improves survival in EGFR-positive cancer patients . TKIs arK-RAS or in PI3K can activate a signaling arm of EGFR regardless of EGFR activation status, rendering the EGFR inhibitor less effective. Also, epithelial to mesenchymal transition (EMT) has also been shown to frequently accompany the acquisition of resistance to TKIs, although the mechanism of the transition is still under investigation [In addition to the challenge of implementing bio-markers for predicting response or resistance to anti-EGFR therapy, another challenge faced by the use of EGFR inhibitors is that the duration of the response is often limited and many tumours invariably develop resistance . Secondatigation ,395,396.tigation ,398. Othtigation ,400,401.KIP1 activity [Inhibition of EGFR mostly causes cell cycle arrest in G1 prior to DNA synthesis ,403,404.activity ,410,411.activity ).TKIs inhibit EGFR phosphorylation, which effectively downregulate ERK and AKT phosphorylations . Many ca3 to compensate for a loss of downstream signaling. Therefore, simultaneous treatment with an RTK inhibitor an PI3K-AKT-mTOR inhibitor has been used, with more favorable responses in vitro and in mice [Therapeutic inhibition of the PI3K-AKT-mTOR pathway has been of intense study, and many potential inhibitors are currently under investigation for cancer. No PI3K or AKT inhibitors have reached the bedside so far, although three mTOR inhibitors have been approved by the FDA . It has  in mice ,432. mTO in mice . Importa in mice . Further in mice .The biological response of EGFR signaling is mediated by the activation of transcription factors. An important role of these transcription factors is to induce the production of CYCLIN D1, which forms a complex with CDK4/6 to initiate cell cycle progression. This EGFR-driven process of chronic proliferation represents one of the important facets behind EGFR-mediated oncogenesis. In this section, we will examine the mechanisms by which the EGFR initiates cell cycle progression, including its upregulation of CYCLIN D, and downregulation of cyclin-dependent kinase inhibitor proteins (CDKis). We will also examine the rationale for combining CDK4/6 and EGFR inhibitors, as this combination has been of recent interest .CYCLIN E and CYCLIN A [The best described role for the EGFR is to drive cells through G1, largely by inducing the activation of the CDK4/6-CYCLIN D complex. A pulse of EGF stimulation during early G1 and another at late G1 are sufficient to drive cells past the restriction point (R point), the instant at which the cell commits to the completion of the cell cycle ,438,439.CYCLIN A ,447. CYCCYCLIN A . ConstitCYCLIN D1 promoter for transcription. Sustained ERK activation is required for progression of G1 into S, as transient ERK activation due to insufficient doses of growth factor does not lead to S-phase progression [The ERK pathway induces CYCLIN D1 expression through its activation of the AP-1 complex. Transcriptionally, ERK induces the expression of immediate early genes (IEGs), including c-FOS and c-JUN . Post-trgression . This hagression .c-FOS [CYCLIN D1 expression [CYCLIN D1 expression [CYCLIN D1 promoter [CYCLIN D1 and the stabilization of both CYCLIN D1 and CYCLIN E [CYCLIN D1 mRNA translation as a result of 4E-BP1 inhibition, with the activation of eIF4E also being important [Concomitant PI3K-AKT signaling also upregulates Cyclin D1 activity. AKT signaling induces the transcription of c-FOS ,451. Actpression . Howeverpression ,453. AKTpression . In the pression . Secondlpromoter . In respCYCLIN E ,458. Lasmportant . The actmportant ,460.Since CYCLIN D activates CDK4/6 to drive G1 progression, selective CDK4/6 inhibitors have been of intense recent research, driven by the availability of highly specific CDK4/6 inhibitors that also exhibit low patient toxicity and high tolerability ,462,463.KIP1 activity [KIP1 inhibits CYCLIN D1-CDK4/6 activity, thereby preventing cells from progressing to S phase [KIP1 also inhibits CYCLIN E-CDK2, further inhibiting exit from G1 [KIP1 has also been reported to induce apoptosis, although the mechanism has not been described [KIP1 also downregulates EGFR transcription [The inhibition of EGFR in various cancers with EGFR inhibitors, including monoclonal antibodies or tyrosine kinase inhibitors, lead to G1/S arrest. Multiple lines of research suggest that the cause of EGFR inhibitor-induced G1/S arrest is the upregulation of p27activity ,410,411. S phase . Active  from G1 . p27KIP1escribed ,469. Intcription ,471. As KIP1 down-regulation [KIP1 and promotes its degradation [KIP1 [KIP1 down-regulation. c-MYC, a commonly-mutated downstream effector of ERK, both activates CYCLIN D1 transcription and inhibits p21CIP1 and p27KIP1 activity, demonstrating the central role of c-MYC in G1/S progression [Many studies have shown that ERK1/2 contributes to p27gulation ,474,475.on [KIP1 , which mgression ,477.CIP1/WAF1 and p27KIP1, by phosphorylation at Thr145 and, Thr157 and Thr198 respectively [KIP1 and p21WAF1 [KIP1 levels, as GSK-3β normally represses p27KIP1 activity by both inhibiting KIP1p27 gene transcription and its protein interaction with CDK2 [Active AKT inhibits CDKis p21ectively ,479. AKT p21WAF1 ,481. Furith CDK2 .de novo upregulation of EGFR signaling pathways (As the ERK and AKT pathways are inhibited by EGFR inhibitors, the subsequent upregulation of CDKis may provide synergy with CDK4/6 inhibitors to provide stronger G1/S arrest. In addition, the inhibition of these EGFR pathways allows the activation of pro-apoptotic proteins, including BIM, CASPASE-9, and BAD ,278,279.pathways , as thesThe EGFR is at the head of one of the most important signaling pathways to mammalian cell physiology, and to oncogenesis. As this review has shown, tremendous amounts of research has been conducted on each of the many components of the EGFR. The sum of our knowledge from this vast array of research is allowing us to create even more effective drugs or drug combinations to target cancers with EGFR aberrations. In addition to understanding these signaling pathways in cancer, the spatial and temporal regulation of the EGFR remain understudied. For example, spatially, EGFR internalization by endocytosis results in EGFR signal attenuation, and has been studied by many groups ,485,486.


Here, extracellular stimuli operate on a wide array of channels, pumps, and ion exchangers to redistribute intracellular Ca2+ among several compartments. The resulting highly defined spatial and temporal patterns of Ca2+ movement can be used to elicit specific cellular responses, including cell proliferation, migration, or death. Plasma membrane (PM) also can directly contact mitochondria and endoplasmic reticulum (ER) through caveolae, small invaginations of the PM that ensure inter-organelle contacts, and can contribute to the regulation of numerous cellular functions through scaffolding proteins such as caveolins. PM and ER organize specialized junctions. Here, many components of the receptor-dependent Ca2+ signals are clustered, including the ORAI1-stromal interaction molecule 1 complex. This complex constitutes a primary mechanism for Ca2+ entry into non-excitable cells, modulated by intracellular Ca2+. Several contact sites between the ER and mitochondria, termed mitochondria-associated membranes, show a very complex and specialized structure and host a wide number of proteins that regulate Ca2+ transfer. In this review, we summarize current knowledge of the particular action of several oncogenes and tumor suppressors at these specialized check points and analyze anti-cancer therapies that specifically target Ca2+ flow at the inter-organelle contacts to alter the metabolism and fate of the cancer cell.Inter-organelle membrane contact sites are emerging as major sites for the regulation of intracellular Ca In card isoform , while Vpoptosis .2+-uptake activity  in B-cell follicular lymphomas , is upreactivity , 69. As nt at ER –72. The n kinase  are amon2+ release from IP3Rs after its phosphorylation by Akt, without affecting histamine-induced Ca2+ release or Ca2+ content  participated to complexes with the protein kinase Akt and protein phosphatase 2a (PP2a), and its binding was critical for Akt- and PP2a-dependent downregulation of IP3Rs phosphorylation. In fact, the amounts of phosphorylated IP3R3 were higher in PML−/− than in PMLhibition . Consisthibition .2+ accumulation and promoted apoptosis increasing SERCA activity, but oncogenic p53 mutants failed to stimulate it. p53 was reported to tether PML , it was possible to develope mipsagargin (G-202). G-202 is an inactive non-toxic prodrug that is activated only in PSMA-expressing epithelial cells, and in tumor vasculature, giving a high precision in tumor killing, specific to hit prostate, and other cancer cells (Drugs containing metal compounds can modify Cal tumors . For exaer cells . G-202 ier cells . The syner cells . In facter cells , while cer cells . Bortezoer cells . Velcadeer cells .2+ uptake in cancer cells after suppression of SERCA activity at the MAMs, while healthy cells remained unaffected (2+ release and consequent apoptosis (Another anti-cancer molecule is resveratrol, which selectively increases mitochondrial Caaffected , 97. Recpoptosis . A modifpoptosis  and diffpoptosis . Multiplpoptosis , 102.2+ overload at mitochondria and improving cisplatin’s toxic effect in human ovarian cancer cells (Another example of a BH3 mimetic involved in calcium remodeling is ABT-737, a non-selective Bcl-2/Bcl-XL inhibitor , 104; iter cells . Recentler cells , 106.2+ flow inside the cell, in addition to their well-known action on signal transduction pathways or on nuclear activities. The hierarchy of the Ca2+ transfer process involves several junctions in the highly compartmentalized cell interior, which warrant communication among different membrane systems. These junctions offer asylum to proteins that fill the role of oncogenes or tumor suppressors during cancer transformation, regulating the Ca2+ concentration inside the mitochondria and reticulum and consequently, altering cell metabolism, preventing apoptosis, and inducing cell migration. Specific therapies can target these junction complexes and revert Ca2+ flux to pro-apoptotic levels to sustain chemotherapy and other cancer therapies. These findings highlight the fundamental role of these inter-organelle junctions as hotspot domains, having pivotal, though not fully understood, roles in the regulation of cancer onset, and progression. Improving our knowledge of the regulation of Ca2+ transfer among these organelles will impact the search for new and more precise treatments for cancer.A growing number of findings indicate that several tumor suppressors and oncogenes can affect several levels of mechanisms regulating CaConception: GP, AR, MP, and PP. Design: GP, AR, LS, CG, MW, MP, and PP. Analysis and interpretation: GP, AR, LS, and CG. Drafting the manuscript for important intellectual content: GP, AR, LS, CG, MW, MP, and PP.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Carica papaya).Ripening affects the quality and nutritional contents of fleshy fruits and is a crucial process of fruit development. Although several studies have suggested that ubiquitin-conjugating enzyme (E2s or UBC enzymes) are involved in the regulation of fruit ripening, little is known about the function of E2s in papaya (CpUBC) genes and found that both exon-intron junctions and sequence motifs were highly conserved among the phylogenetic subgroups. Using real-time PCR analysis, we also found that all the CpUBC genes were expressed in roots, stems, leaves, male and female flowers, and mature fruit, although the expression of some of the genes was increased or decreased in one or several specific organs. We also found that the expression of 13 and two CpUBC genes were incresesd or decreased during one and two ripening stages, respectively. Expression analyses indicates possible E2s playing a more significant role in fruit ripening for further studies.In the present study, we searched the papaya genome and identified 34 putative UBC genes, which were clustered into 17 phylogenetic subgroups. We also analyzed the nucleotide sequences of the papaya UBC (To the best of our knowledge, this is the first reported genome-wide analysis of the papaya UBC gene family, and the results will facilitate further investigation of the roles of UBC genes in fruit ripening and will aide in the functional validation of UBC genes in papaya. Carica papaya) is an economically important fruit crop that is widely grown in tropical and sub-tropical regions, and its fleshy fruits play an important role in the human food supply, as well as in human nutrition and health [bananas, oranges), and mango [Papaya  has long served as an excellent model for studies of fleshy fruit development and ripening [rin, which encodes a MADS-box transcription factor [Nr, which encodes an ethylene receptor [Cnr (Colorless nonripening), which encodes an SBP-box transcription factor [Gr (Green ripe), which encodes a still poorly defined component of the ethylene transduction pathway [SINAC4, a new tomato NAC transcription factor that positively regulates fruit ripening and carotenoid accumulation [APETALA2a (AP2a), a transcription factor that regulates fruit ripening via regulation of ethylene biosynthesis and signaling [LeNCED1, which initiates abscisic acid (ABA) biosynthesis at the onset of fruit ripening and might act as an original inducer in tomato [SHATTERPROOF, TAGL1 [TDR4/FUL1, and MBP7/FUL2 [C. papaya.Ethylene, in particular, has been extensively studied, owing to its crucial role in the ripening of climacteric fruits –9, and tripening , 12, andn factor ; Nr, whireceptor ; Cnr , 20 in Caenorhabditis elegans [Arabidopsis thaliana [Ubiquitination is an essential cellular process in all eukaryotes, from unicellular yeast to humans, and the ubiquitination-proteasomal pathway has been implicated in diverse aspects of eukaryotic cellular regulation, owing to its ability to degrade intracellular proteins , 22. Therevisiae , 12, 18, elegans , 37 in b elegans  and Arabthaliana , 48, 52,thaliana –31.A. thaliana have been implicated specifically [A. thaliana ubiquitin-conjugating gene AtUBC13 has been implicated in epidermal cell differentiation and iron deficiency responses [OsUBCs were differentially expressed under drought and salt stress conditions [Zea mays, 48 and 16 ZmUBC genes were significantly upregulated under salt and drought stress conditions, respectively [Previous studies have suggested that ubiquitination plays an important role in plant growth, development, and tolerance against various abiotic stresses, and several of the UBC genes in ifically , 33. Foresponses , 35, andesponses , whereasesponses . In ricenditions , and in ectively .SlUBC32, which is upregulated during tomato fruit ripening and downregulated in the rin mutant, plays an important role in the regulation of fruit ripening [MaUBC genes are reportedly up- and downregulated during different ripening stages [C. papaya plant tissues and at different stages of fruit ripening. To the best of our knowledge, this is the first reported genome-wide analysis of the papaya UBC gene family, and the results will facilitate further investigation of the roles of the UBC genes in fruit ripening and will aide in the functional validation of UBC genes in papaya.Several studies suggest that E2s also participate in the fruit ripening process. In tomato, for example, ripening ; and in g stages . HoweverC. papaya cv. ‘sun up’) plants were obtained from the South Subtropical Crops Research Institute of the Chinese Academy of Tropical Agricultural Science , and root, stem, leaf, male flower, female flower, and fruit (mature green) organs were collected separately. For the fruit ripening experiment, we also collected fruit pulp from four development stages: immature green (IG), mature green (MG), breaker (Br), and mature fruit (MF). The experiments were performed in triplicate, and all samples were immediately frozen in liquid nitrogen and stored at −80°C for expression analysis.Papaya (Arabidopsis and yeast (S. cerevisiae) UBC protein sequences were retrieved and used as queries in BLASTP searches against the C. papaya Genomics Database  and PLAZA 3.0 (http://bioinformatics.psb.ugent.be/plaza/versions/plaza/) [Arabidopsis, S. cerevisiae, and S. lycopersicum were downloaded from the Arabidopsis Information Resource (http://www.arabidopsis.org/), Saccharomyces Genome Database (http://www.yeastgenome.org/), and Sol Genomics Network Tomato Database (http://solgenomics.net) websites, respectively. All putative papaya UBC protein sequences were then subject to domain analysis using Simple Modular Architecture Research Tool  [http://expasy.org/tools/) [http://gsds.cbi.pku.edu.cn/) [To identify potential members of the papaya UBC protein family, published /plaza/) . Amino aerg.de/) , and inferg.de/) . The mol/tools/) . Finallyedu.cn/) .S. cerevisiae, 48 UBC proteins from Arabidopsis thaliana, and 52 UBC proteins from S. lycopersicum were subject to multiple sequence alignment, using MUSCLE [http://www.megasoftware.net/), and the significance of nodes was assessed using a bootstrap test with 1,000 iterations [http://taxonomy.zoology.gla.ac.uk/rod/treeview).To evaluate the evolutionary relationship of the CpUBC proteins, full-length amino acid sequences of 34 CpUBC proteins, 15 UBC proteins from g MUSCLE , with deerations . Represe(-ΔCt) method. The analysis included cDNA from the three biological samples for each tissue, and all the reactions were run in triplicate. In the comparative expression analysis of CpUBCs, genes that were up- or downregulated by at least two-fold were considered differentially expressed.Total RNA was extracted for cDNA synthesis using the Super fast new plants of RNA extraction kit , following the manufacturer's instructions. Reverse transcription reactions were performed using the PrimeScript RT reagent kit with gDNA Eraser , according to the supplier’s manual. Real-time PCR was performed using a Bio-Rad real-time thermal cycling system  and SYBR-green to assess the expression levels of 34 candidate CpUBC genes. The gene-specific primers were designed according to the CpUBC gene sequences using Primer 5 software and checked using Blast  In additArabidopsis and yeast (S. cerevisiae) UBC protein sequences as queries in BLASTP searches against the CpGDB and PLAZA 3.0[CpUBC1 to CpUBC34. In these 34 CpUBCs, two were identified as Related to Ubiqutin (RUB) conjugating enzymes (CpUBC18 and CpUBC29), and one was identified as a SUMO-conjugating enzyme (CpUBC11). Six other UBC proteins (CpUBC2/14/16/19/24/25) lacked the Cys active site, identifying them as ubiquitin-conjugating enzyme variants (UEVs), which are not active by themselves, thus leaving 25 potential ubiquitin E2s. To better understand the papaya E2s, we constructed a recombinant E2 protein library of all 34 CpUBCs. The predicted proteins ranged from 98 (CpUBC15) to 668 amino acids (CpUBC20) in length, with corresponding molecular masses of 10.74 kDa and 74.03 kDa, respectively, and predicted isoelectric points of between 4.29 (CpUBC33) and 9.64 (CpUBC27).In the present study, we used the published PLAZA 3.0, a totalCpUBC8, CpUBC14 and CpUBC19) to seven (CpUBC20 and CpUBC23), with most of CpUBC genes containing three to five introns (n = 76.5%)  . In addihttp://meme-suite.org/index.html). The results indicated that the highly conserved sequence of the UBC domain is HPNINSNGSICLDILKEQWSP  located at the C-terminal side of the active cysteine was conserved in most of the CpUBC genes . The conILKEQWSP .According to the UBC domain and the N- or C-terminal structure, E2 proteins are divided into four types. Class I E2s contain only the catalytic domain; Class II E2s contain N-terminal extensions; Class III E2s contain C-terminal extensions; and Class IV E2s have both N- and C-extensions , 46. In S. cerevisiae proteins or proteins contained within the group ; however, there were no yeast proteins in groups UBC14, UBC15, UBC16, and UBC19, which indicated that the groups may be plant-specific or were lost in yeasts. Interestingly, the subfamilies UBC4/5 or UBC3/7 shared two highly identical paralogous yeast genes, respectively. For example, during the phylogenetic analysis of CpUBC genes using MEGA 5.0, UBC4 and UBC5 always clustered into the same subgroup, as did UBC3 and UBC7. According to previous reports [Furthermore, phylogenetic analysis revealed that the UBC proteins could be divided into 14 E2 groups and three independent UEV groups , 11 (CpUBC2/3/15/21/23/27/28/29/30/31/33), and seven (CpUBC7/8/11/12/14/16/20) genes were lowest in leaves, roots, and stems, respectively, and that the expression of only one gene was lowest in the male flowers (CpUBC32) and female flowers (CpUBC19), respectively. Meanwhile, 19 (CpUBC1/2/3/5/ 6/9/10/11/12/15/17/20/23/24/26/30/31/33/34) and two genes (CpUBC21 and CpUBC22) were highly expressed in male flowers and female flowers, respectively, which implicated their involvement in the development of floral sex organs. Four (CpUBC25/27/28/29), five (CpUBC7/8/14/16/19), and two (CpUBC4 and CpUBC32) genes were highly expressed in fruits, leaves, and stems, respectively, and a single gene (CpUBC13) was highly expressed in roots. Therefore, our results suggested that CpUBC genes play multiple roles in papaya development.All 34 of the candidate CpUBC genes were differentially expressed among the six papaya organs . In addiCpUBC4/6/7/8/9/11/12/14/16/19/20/28/34) and two (CpUBC2 and CpUBC10) of the genes showed higher or lower expression during the progress of papaya fruit ripening, respectively, whereas the expression of the remaining genes was not significantly different (CpUBC7/8/9/11/12/14/19/20) were slightly increased at the IG, MG, and Br stages and most highly expressed at the MF stage. Meanwhile, the expression of four genes (CpUBC6/16/28/34) continuously increased during the first three stages of fruit ripening, before being decreased at the MF stage, and the expression of CpUBC4 was increased at MG stage and then reduced at the two late stages. These results suggest that the papaya E2s maybe playing a significant role in fruit development and ripening.In the present study, we also analyzed the expression of the 34 CpUBC genes at different fruit ripening stages. We found that 13  proteins , one SUMO-conjugating enzyme (ScUBC12), and two UEV protein  [Arabidopsis, of which two are RUB-conjugating enzymes , one is a SUMO-conjugating enzyme , and eight others are UEVs, thus leaving 37 potential E2s [Arabidopsis [Arabidopsis, rice, tomato, maize, and banana are ~125, ~466, ~900, ~2300, and 523 Mb, respectively. However, although the genome sizes of rice and tomato are 3.7 and 7.2 times larger than that of Arabidopsis, respectively, the genomes of the three species contain a similar number of UBC genes ; and although the genome size of maize is 4.3 times larger than that of banana, the genomes of these two species also contain a similar number of UBC genes . Therefore, the difference in number of UBC genes among these species cannot be explained by genome size.In the present study, we identified genes encoding 34 proteins , which i_009919) . In additial E2s . Among 4tial E2s ; and of tial E2s . The numbidopsis , rice [2bidopsis , maize [bidopsis , and banbidopsis , respectAlternatively, gene duplication events have been demonstrated to play a significant role in the expansion of gene family members in genomes , and an Arabidopsis, rice, and maize, the UEV subgroups were taken into account [Through systematic phylogenetic analyses, the present study provides a detailed classification and nomenclature of papaya E2s. The phylogenetic analysis categorized the 34 CpUBCs into 17 discrete groups, which was similar to the number of groups described in other species, such as tomato, maize, and banana –31. Howe account . In our  account , banana  account , and pap account , 30, werArabidopsis, and previous research has shown that the genomes of maize and rice each contain eight and three members of the UBC9 and UBC12 groups, respectively [Arabidopsis and papaya for which no probable homologs exist in budding yeast; however, all three groups have potential homologs in animals [In addition, the number of UBC genes was obviously different among the groups. The largest groups (UBC4/5) included eight members, whereas the UBC1, UBC2, UBC6, UBC9, UBC10, UBC13, UBC14, and UBC15 groups were only represented by one member each. The UBC4/5 groups was also the largest in other species, which indicates that, in plants, the group may possess more diverse functions than other groups. Phylogenetic data also suggested that the UBC9 and UBC12 groups were expanded in monocots but not in ectively , 30. How animals , which iArabidopsis, for example, AtUBC1 and AtUBC2 are ubiquitously expressed in roots, leaves, flowers, and seedlings, and the double mutant atubc1-1atubc2-1 exhibits a dramatically reduced number of rosette leaves and an early-flowering phenotype [MaUBC10/11/33/34/61 are highly expressed in most organs, especially in roots, stems, leaves, and MaUBC6/11/34/35/45/61 were highly expressed in stems. In addition, 12 MaUBC genes (MaUBC13/18/29/33/34/36/43/46/48/53/67/70) were predominantly expressed in roots [Although the genome sequence of papaya has been reported, the identification and functional studies of papaya genes have proceeded at a slow pace. Analysis of the temporal and spatial expression patterns of genes may provide useful information for characterizing their functions. Previous studies have shown that UBC genes have different expression patterns in different organs. In henotype . In banaOgUBC1 from wild rice is involved in cellular responses to biotic and abiotic stresses [OsUBC2/5/18) and five UBC genes (AtUBC13/17/20/26/31) from rice and Arabidopsis, respectively, are significantly reduced in response to salt and drought stress, whereas only three rice genes (OsUBC13/15/45) are significantly upregulated [SlUBC6/8/24/32/41/42) were directly regulated by the fruit-ripening regulator RIN, which suggests that specific UBC genes might be involved in fruit ripening, and in banana, five UBC genes (MaUBC1/9/70/68/71) exhibited approximately 10- to 40-fold higher levels of expression at the fifth stage than at other stages of fruit ripening, whereas seven other UBC genes (MaUBC8/16/17/33/34/56/61) exhibited continuously increasing expression throughout the fruit development. In the present study, 13 (CpUBC4/6/7/8/9/11/12/14/16/19/20/28/34) and two (CpUBC2 and CpUBC10) of the 34 CpUBC genes were up- or downregulated during the progression of papaya fruit ripening, respectively  are critically important in many aspects of plant growth and development, as well as in physiological processes, such as stress responses . For exastresses , and thregulated . Althougegulated , 31. In ectively , and ourIn the present study, we describe the genome-wide identification and analysis of UBC genes in papaya. A total of 34 putative CpUBC genes were identified, and phylogenetic analysis indicated that genes could be divided into 17 subfamilies. Analysis of exon-intron junctions and sequence motifs revealed high levels of conservation within and between phylogenetic groups. In addition, all the CpUBC genes were detected in roots, stems, leaves, male and female flowers, and mature fruit, although some genes showed higher or lower expression in one or several specific organs. Similar to the results in tomato and banana, we also found that the expression of 13 and two CpUBC genes were incresesd or decreased during one and two ripening stages, respectively. Therefore, the results of the present study suggest that CpUBC genes are involved in the regulation of fruit development and ripening processes. The results also provide novel insights into the function of plant UBC genes and will facilitate further investigation of the roles of the UBC genes in fruit ripening.S1 Table(DOC)Click here for additional data file.


While functional MRI and PET studies have shown altered task-related brain activity in PKAN, we want to find such differences also in the resting state (RS).Here we used ICA based analysis to investigate RS fMRI data to compare connectivity of 11 well known networks , Exectutive Control, Fronto-parietal 1, Fronto-parietal 2, Salience, Sensorimotor, Visual1, Visual2, Visual3 network) between patients with PKAN and healthy controls suggesting deficits in related neuropsychological functions.We obtained RS fMRI series  in 17 PKAN patients but 3 were discarded because of excessive movement, (mean age 17.2a±7.1) on stable medication and 15 healthy controls (22.5a±8.3).Subjects were asked to lie in the scanner keeping eyes closed with no further specific instructions. Data were pre-processed; we applied FSL MELODIC (pICA) yielding IC, we used FIX to auto-classify ICA components which represent artifacts and an automated routine to select for each subject the component matching the anatomical definition of resting state networks.SPM12 was used for second level analysis, we used two sample t-test to compare networks functional connectivity between groups. In addition, we used multiple regression to correlate RS networks activity components with Dystonia score.Our method reliably identified all networks in every control and patients. We found significant differences in the anatomical pattern of areas. Patients showed decreased functional connectivity in comparison to healthy controls in portions of Fronto-parietal 1, Fronto-parietal 2 and Visual1 networks; in addition, patients showed increased functional connectivity in comparison to healthy controls in portions of Cerebellum, DMN, Executive Control, Salience and Visual1 networks. Finally, significant correlation was found between dystonia score and functional connectivity of Cerebellum, Fronto-parietal1, Fronto-parietal2, Salience, Sensorimotor and Visual2 networks.Well known resting state networks were reliable identified from RS fMRI in PKAN patients. The differences in anatomical distribution point to possible alterations in functional connectivity in PKAN, which suggests disruption in cerebellum, DMN, fronto-parietal, salience and visual activity. Correlations with dystonia suggest a direct relation to motor items, which would support a clinical significance of altered RS networks activity.


Eosinophils are typically considered to be specialized effector cells that are recruited to the tissues as a result of T helper type 2 (Th2) cell responses associated with helminth infections or allergic diseases such as asthma. Once at the site of injury, eosinophils release their cytotoxic granule proteins as well as preformed cytokines and lipid mediators, contributing to parasite destruction but also to exacerbation of inflammation and tissue damage. Accumulating evidence indicates that, besides their roles in Th2 responses, eosinophils also regulate homeostatic processes at steady state, thereby challenging the exclusive paradigm of the eosinophil as a destructive and inflammatory cell. Indeed, under baseline conditions, eosinophils rapidly leave the bloodstream to enter tissues, mainly the gastrointestinal tract, lungs, adipose tissue, thymus, uterus, and mammary glands, where they regulate a variety of important biological functions, such as immunoregulation, control of glucose homeostasis, protection against obesity, regulation of mammary gland development, and preparation of the uterus for pregnancy. This article provides an overview of the characteristics and functions of these homeostatic eosinophils. Eosinophils have long been perceived as terminally differentiated cytotoxic and destructive cells that play an effector role mainly in helminthic infections and allergic reactions, such as asthma . Howeverlamina propria of the small intestine (5 to 2 × 106 cells)  , 9. Pulm5 to 2 × 6 cells  in the circulation , a chemokine produced by local cells such as epithelial cells, endothelial cells, fibroblasts, and monocytes –23. CorrhiCD62L−CD101hi cells with a segmented nucleus (CD101 being an iEos marker that is not expressed by lung hEos) (+CD62L+IL-3Rlo cells) are phenotypically distinct from the iEos isolated from the sputa of eosinophilic asthmatic patients (Siglec-8+CD62LloIL-3Rhi cells), confirming the mouse findings  and combined expression of CCR3, Siglec-F, and CD125  , 9, 34 , 17, 35.Gata1 promoter has been genetically deleted, specifically lack eosinophils, including blood and tissue hEos , CCAAT/enhancer-binding protein-α and -ε (C/EBP-α and -ε), E26 family transcription factor PU.1 (PU.1), and X-box-binding protein-1 , 40–42. sue hEos , 36, 43.sue hEos . Interessue hEos , 44, 45 sue hEos  than thesue hEos –48.Depending on the type of tissue they infiltrate, hEos are fulfilling completely different tasks, suggesting the local environment is able to drive hEos functions according to its specific needs. Here, we will review the tissue-specific homeostatic functions of hEos, also summarized in Figure In vitro, Chen and colleagues have shown that small intestinal hEos are able to induce differentiation of naive T cells into Foxp3+ regulatory T cells through IL-1β- and retinoic acid-dependent mechanisms A-producing plasma cells , 35, conchanisms . More ret IL-1Rα . AltogetNippostrongylus brasiliensis, which triggered a greater eosinophil recruitment in the adipose tissues, exhibit a long-lasting improved sensitivity to insulin and glucose tolerance , and such hEos produce IL-4, thereby favoring the polarization of adipose macrophages toward the alternatively activated phenotype . AAMs plolerance .This important hEos/macrophage axis is regulated by ILC2s, which sustain adipose hEos and AAMs , and is It is known for decades that hEos infiltrate the non-pregnant uterus of rodents and humans in a cyclic manner, with a peak during estrus , 57, 58,The presence of thymic hEos in the close vicinity of immature double-negative thymocytes and their abundance in neonates suggest that they may contribute to the process of central tolerance and negative T-cell selection . SupportA role for eosinophils in regulating postnatal mammary gland development has been proposed in mice . Indeed,Anxa1, Nedd4, Runx3, Serpinb1a, and Ldlr, that are implicated in the maintenance of lung immune homeostasis, and especially in the negative regulation of Th2 cell responses (Microarray analyses revealed that lung hEos, unlike lung iEos, express several genes, such as esponses . In lineesponses , confirmesponses .Although hEos are far from being fully characterized, it is fascinating to see how fast our understanding of the complexity of their phenotype and functions is growing. The fact that these cells exert crucial homeostatic roles at multiple levels merits further investigations and is of medical importance. Indeed, anti-eotaxin-1 and eosinophil-depleting agents, such as humanized anti-IL-5 receptor antibodies and anti-Siglec-8 molecules, are currently being developed to treat eosinophilic disorders such as allergic asthma , 61–66, TM, CM, and FB conducted a review of the literature. TM and FB wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Among a great variety of cell surface receptors, the largest superfamily is G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors. GPCRs can modulate diverse signal-transduction pathways through G protein-dependent or independent pathways which involve β-arrestins, G protein receptor kinases (GRKs), ion channels, or Src kinases under physiological and pathological conditions. Recent studies have revealed the crucial role of GPCRs in the tumorigenesis and the development of cancer metastasis. We will sum up the functions of GPCRs—particularly those coupled to chemokines, prostaglandin, lysophosphatidic acid, endothelin, catecholamine, and angiotensin—in the proliferation, invasion, metastasis, and angiogenesis of hepatoma cells and the development of hepatocellular carcinoma (HCC) in this review. We also highlight the potential avenues of GPCR-based therapeutics for HCC. Hepatocellular carcinoma (HCC) is the second most prevalent cause of mortality and poor-prognosis malignancy characterized by recurrence after surgery and metastasis worldwide . While sB) receptors, a class of eight metabotropic glutamate receptors, and a number of orphan receptors. Class D chemically communicates in some of the organisms. Class E is used to form the chemotactic signaling system of slime molds. Finally, class F is essential for Wnt binding [GPCRs, which are vitally important eukaryotic signal transduction transmembrane proteins, constitute the largest protein family, with over 800 members in the proteome of humans. They have a common structure of seven transmembrane helices (H1–H7) joined with three intracellular  and three extracellular  loops, an extracellular N-term, and an intracellular C-term. In addition, according to sequence and structure similarity, GPCRs were usually grouped into six main families: class A (rhodopsin-like), class B (secretin receptor family), class C (metabotropic glutamate/pheromone), class D , class E (cyclic adenosine monophosphate (cAMP) receptors), and class F (frizzled/smoothened) [ binding .The classical pathway through which GPCRs mediate cellular physiology is that the bundle with ligands induces conformational alteration in the transmembrane and intracellular domains of the receptor, thus communicating with heterotrimeric G proteins that consist of the Gα, Gβ, and Gγ subunits. Activated GPCRs catalyze the liberation of guanosine diphosphate (GDP) and then bundle with guanosine triphosphate (GTP) for G protein activation. Afterwards, both the Gα-GTP and Gβγ subunits lead to various signaling cascades and different downstream effector proteins to regulate large quantities of cellular physiology .GPCRs transduce signals through their direct interactions with β-arrestins. GPCRs scaffold many intracellular signaling proteins by different signaling pathways and induce signaling cascades independent of G protein activation—including Wnt, Notch, transforming growth factor-β (TGF-β), and the hedgehog (Hh) pathways—as well as downstream kinases—such as phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) . AdditioFurther, GPCRs can utilize receptor tyrosine kinases (RTKs) to mediate important cellular responses, such as proliferation, differentiation, and survival . DependiIncreasing clinical and experimental data have suggested that GPCRs play a vital but not fully understood role in HCC progression and metastasis by modulating diverse downstream signaling pathways. Furthermore, we have learned that GPCRs—particularly those coupled to chemokines, prostaglandin, lysophosphatidic acid, endothelin, catecholamine, and angiotensin—have a central function in proliferation, invasion, metastasis, and neovascularization of hepatoma cell and the development of HCC, which are summarized in Chemokines, which are small proinflammatory chemoattractant cytokines, couple to GPCRs to induce conformational alteration and trigger intracellular signaling pathways containing cell movement and activation . To dateN-nitrosodiethylamine (DEN)-induced HCC, and HCC induced by hepatitis B virus surface (HBs) antigen-primed splenocyte transfer to myelo-ablated syngeneic HBs antigen transgenic mice. CCL2, CCL3, and CCL15—cognate ligands of CCR1—markedly increase in endothelial cells from HCC tissues. Furthermore, proliferation of endothelial cells also significantly enhances and directly modulates vessel remodeling via bundling with CCR1 in an autocrine way [The CC subclass is the largest group in all chemokine receptors that has a significant influence on HCC. CCR1 is aberrantly overexpressed in human HCC tissues, rine way ,20. Furtrine way . Furtherrine way . In addirine way .CCR5 and its ligand CCL3, which regulates inflammation, are crucial in the microenvironment of HCC, recruiting packs of macrophages and neutrophils into the inflammation sites in different HCC cell lines when stimulated with IL-1α or IL-1β .+CD62L−CCR7− effector memory phenotype is shown [CCR6 is overexpressed in the intrahepatic metastasis of HCC. Angiogenesis benefits from the accumulation of intratumoral IL-17-producing cells in the microenvironment of HCC. It is due to these cells that CCR6 is highly expressed and a CD45ROis shown . Howeveris shown . Thus, C+ and CD8+ T cells, dendritic cells (DCs), interleukin-12 (IL-12), and interferon-γ (IFN-γ) is dramatically increased. Consequently, microvessels are reduced during the angiogenesis of HCC [High expression of CCR7 increases intrahepatic and lymphatic HCC dissemination . Howevers of HCC .CCR9 is strikingly increased in HCC tissue samples. Overexpression of CCR9 is associated with increased tumor nodes, vascular invasion, and a high Edmondson–Steiner grade. Furthermore, ectopic expression of CCR9 increases the proliferative ability and tumorigenicity of HCC cells. However, blocking CCR9 mediates deterioration of HCC cells by decreasing the cell-cycle regulators p21 and p27, as well as increasing cyclin D1 . This stAnother major part of chemokines is composed of the CXC subclass in HCC. Compared with adjacent and normal liver tissues, the level of CXCR2 is significantly enhanced in HCC, which accelerates invasion and metastasis . The CXC+ liver cancer cells [Moreover, the CXCL9/CXCL10-CXCR3 axis has a significant impact on HCC patients’ prognosis. Overexpression of CXCR3 is frequently associated with tumor size, tumor differentiation, portal invasion, and metastasis in HCC tissues via increasing levels of ERK1/2 phosphorylation in the MAPK signaling pathway, then increasing the MMP-2 and MMP-9, and accordingly accelerating invasion and migration of CD133er cells . It has The angiogenesis, invasion, and tumor growth of human HCC cells are correlated with the overexpression of CXCR4 or CXCR7. Exposure to CXCL12 regulates a perinuclear translocation of CXCR4 in Huh7/Hep3B cells and promotes the invasion of Huh7 cells. Also, the high level of CXCR4 in HCC patients is markedly associated with advanced local tumors, lymphatic metastasis, distant dissemination, and decreased three-year survival rate ,34,35. TThe level of CXCL16 and CXCR6 are upregulated in tumor tissues and HCC cell lines. This high expression is associated with the increase of neutrophils in tumor tissues, and with a poor prognosis of HCC patients. In addition, triggering the CXCR6 signal pathway in HCC cells leads to the upregulation of IL-6 and IL-8. However, blocking the CXCL16-CXCR6 axis stops this effect. This suggests that the elevated expression of CXCR6 creates a protumor inflammatory environment, promoting HCC invasion .CX3CL1 is the only member of the CX3C subclass. The CX3CL1/CX3CR1 axis contributes to the development of HCC. HCC patient tissues with a high level of both CX3CL1/CX3CR1 have a markedly lower proliferating cell nuclear antigen labeling index, which leads to a better prognosis in terms of overall survival and being disease-free, with fewer intra- and extrahepatic recurrences. These results indicate that CX3CR1 could be regarded as a prognostic factor . CX3CR1 XCL1 is the sole member of the C subclass. X-C motif chemokine receptor1 (XCR1), as the only receptor of XCL1, suppresses liver cancer growth and tumorigenesis (at least in part) through the MAPK and PI3K/Akt signaling pathways. It does, however, promote metastasis via EMT .2+ concentrations. EP2 and EP4 receptors bind to G proteins, signaling via upregulating the level of intracellular cAMP and the activation of protein kinase A (PKA), while EP3 receptors bind to Gαi-proteins and signal by reducing intracellular cAMP. They may influence the growth, invasion, adhesion, metastasis, and angiogenesis of cancer cells, consequently participating in the tumorigenesis and progression of HCC [Prostaglandin E2 (PGE2) exerts its physiological effects by binding cognate prostanoid receptors, which are divided into EP1, EP2, EP3, and EP4. Normally, EP1-receptors bind to Gαq-proteins and trigger the signal pathway through PLC and enhanced intracellular Can of HCC .The level of EP1 receptors is higher in HCC cells compared with normal human hepatocytes. PGE2 couples to the EP1 receptor, a process in which EGFR, Src, and p44/42 MAPK are all involved. Then, p44/42 MAPK activates the mTOR pathway, which in turn upregulates the YB-1 expression, promoting cancer cell invasion through regulating EMT-associated gene expression . Also, tThe EP2 receptor has a vital influence on accelerating Huh-7 cell migration and invasion by triggering the EP2/Src/EGFR, an RTK, mediating the PI3K/Akt/mTOR pathway. mTOR upregulates the expression level of the Snail protein, thus regulating tumor cell proliferation, growth, and survival . MeanwhiLysophosphatidic acid (LPA) is a biological lipid regulator with various physiological and pathological functions on different kinds of cells. Initially, LPA elicits its biological actions through coupling to three subclasses of the endothelial differentiation gene (Edg) family GPCR . Apart from these receptors, there is another group from the Edg family, including LPAR4, LPAR5, and LPAR6. They regulate various cell biological functions such as motility, migration, and proliferation .LPAR1, 3, and 6 mRNA and protein expression is high in the human hepatoma cell line HuH7. Stimulation with LPA promotes HuH7 proliferation and increased motility in dose-dependence . LPA, HCLPAR1 protein is upregulated in HCC compared to normal tissues. Silencing LPAR1 markedly attenuates LPA-induced MMP-9 expression levels and the invasive ability of HCC cell. LPAR1 elevates MMP-9 expression via both PI3K/Akt and protein kinase Cδ (PKCδ)/p38 MAPK pathways . In addiAdrenergic receptors (ARs) appear in a variety of tissues and mainly regulate the sympathetic nervous system (SNS) system and other cellular processes through coupling to catecholamines . However, aberrant adrenergic signaling contributes to the initiation and deterioration of HCC, including cell motility and trafficking, angiogenesis, inflammation, activation of tumor-associated viruses, apoptosis/anoikis, cellular immune response, and EMT. The β2-, α1-, and α2-ARs are the primary subclass in the human liver. The β2-AR bundling with Gs and α2-AR bundling with Gi are related to the biochemical effector-AC-system, which catalyzes cyclic 3′,5′-adenosine monophsphate (cAMP) formation from ATP. Also, the α1-AR bind to Gq/11-protein and activate PLC .α1-AR is lower in HCC tissues compared with those of nonadjacent, noncarcinoma livers. Tumor-induced stress also increases the catecholamine level upon chronic stimulation of the SNS that may result in desensitization and inhibition of the α1-AR-Gq/11-PLC-pathway . HoweverAngiotensin II (Ang II), a key bioactive peptide of the rennin-angiotensin system, mediates its biological effects via coupling to an angiotensin II type 1 receptor (AT1R) or AT2R. Although they belong to the same subclass of GPCRs, their distribution and intracellular signaling pathways are different. Both, however, have a crucial impact on neovascularization and tumor progression. For example, AT1R and AT2R have proangiogenic effects in colorectal cancer liver metastases and melanoma ,65.AT1 induces production of angiogenic factors such as VEGF, Tie-2, and angiopoietin-2 (Ang-2) in MHCC97H cells. Furthermore, VEGF and Ang-2 significantly increase blood vessel formation . AT1R, wSome research suggests that AT2R has a proangiogenic function and works in cooperation with the AT1R subtype to upregulate the level of VEGF and angiogenic tube formation ,69. HoweThe Smo receptors, one of the class F GPCRs, are core components of the classical hedgehog (Hh) signaling pathway during embryogeny and adulthood . It is wUpregulation of the Smo proto-oncogene, and an enhancement in the stoichiometric ratio of Smo to patched (Ptc) mRNA levels, correlates with tumor size, a prognostic monitor in HCC biology . It has GPCRs comprise the largest family of transmembrane receptors, with more than 800 members, in which hundreds of members are classified as orphan GPCRs (oGPCRs), with no identifiable endogenous ligands .GPR137 is elevated in some human HCC cell lines. Depletion of GPR137 via lentivirus mediated RNA interference (RNAi) in HCC cell lines HepG2 and Bel7404 remarkably reduces cell viability and colony-formation ability, which increases cell apoptosis . HoweverHCC is the most stubborn cancer, resistant to chemotherapies for a variety of tumors, and there are too few drugs available for scientific chemotherapy at present. However, recent advances in the understanding of GPCRs and their downstream effectors reveal a large number of novel drug targets for groundbreaking strategies in HCC diagnosis, prevention, and treatment.+ T cell response [For example, blockade of CCL2/CCR2 signalling with a CCR2 antagonist, RDC018, inhibits HCC growth and metastasis, reduces postsurgical recurrence, and enhances survival. Furthermore, it suppresses murine liver tumor growth via inhibiting the recruitment of inflammatory monocytes and infiltration and M2-polarisation of tumorassociated macrophages (TAMs), resulting in a reversal of the immunosuppression status of the tumor microenvironment and activation of an antitumorous CD8response . In addiresponse . This stresponse . Furtherresponse ,87. Thisresponse . ConsequA crosstalk between the PGE2 and EGFR/c-Met signaling pathways affects HCC cell invasive ability. PGE2-induced transactivation of EGFR in HCC cells occurs through activation of c-Src, followed by c-Met activation. Thus, when treating with the EP1 receptor antagonist ONO-8711, the PGE2-mediated phosphorylation of EGFR and cell invasion are inhibited ,89. FurtThe β2-AR antagonist propranolol has an impact on enhancing sorafenib efficacy, since blocking β2-AR signaling results in HIF-1α autophagic degradation .AR) selective inhibitor BQ-123 suppresses ET-1-induced migration and invasion, and downregulates MMP-3 production in HepG2 cells via inhibiting activation of the ERK1/2 and Akt pathways [AR could be a potential target for HCC treatment.Furthermore, the endothelin type A receptor (ETpathways . This shGDC-0449 (vismodegib), an antagonist of Smo, significantly decreases tumor size and cell infiltration of HCC in HCC-bearing mice. Gene expression of the sonic hedgehog (Shh) pathway molecules is altered, such as the upregulated Shh expression and downregulated smoothened expression in tumor areas after treatment with GDC-0449 . These GAlthough GPCRs constitute the largest superfamily of transmembrane receptors participating in signal transmission, in clinical practice, only a few anticancer drugs have been applied to HCC. In addition, the function played by GPCRs and its cognated ligands in HCC development and pathophysiological progression is intricate. Therefore, GPCRs and their downstream-regulated effectors provide many drug targets for novel strategies in HCC prevention and treatment. Consequently, the full knowledge of the transduction crosstalk connecting several GPCR-dependent signals with other transduction pathways will promote further research regarding the biological potential of these receptors, meanwhile providing valuable avenues for the development of novel, GPCR-based therapeutic strategies for cancer diagnosis. Finally, the persistent efforts to fully describe a rich source of GPCRs will certainly lead in the near future to the identification of novel targets toward groundbreaking pharmacological treatment strategies in HCC patients.