Novel Pannexin 1 isoform is increased in cancer.

Pannexin 1 (PANX1) is upregulated in many cancers, where its activity and signalling promote tumorigenic properties. Here, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer and glioblastoma multiforme. This isoform was increased upon hPANX1 CRISPR/Cas9 deletion targeting the first exon near M1, suggesting a potential alternative translation initiation (ATI) site. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at N254, and can interact with both ß-catenin and full length hPANX1. A double deletion of hPANX1 and hPANX1-25K reduces cell growth and viability in cancer cells. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and normal skin, indicating that it may be differentially regulated in normal and cancer cells.

Chemical proteomics reveals the target landscape of 1,000 kinase inhibitors.

Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound-target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology.

Involvement of the AKT Pathway in Resistance to Erlotinib and Cabozantinib in Triple-Negative Breast Cancer Cell Lines.

Resistance to protein tyrosine kinase inhibitors (TKIs) presents a significant challenge in therapeutic target development for cancers such as triple-negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Due to increased expression, the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met are potential targets for treatment. However, targeted anti-EGFR and anti-c-Met therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that adaptive responses in regulatory kinase networks within the EGFR and c-Met signaling axes contribute to the development of acquired erlotinib and cabozantinib resistance. To test this, we developed two separate models for cabozantinib and erlotinib resistance using the MDA-MB-231 and MDA-MB-468 cell lines, respectively. We observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion, and activation of EGFR or c-Met downstream signaling (respectively). Using a SILAC (Stable Isotope Labeling of Amino acids in Cell Culture)-labeled quantitative mass spectrometry proteomics approach, we assessed the effects of erlotinib or cabozantinib resistance on the phosphoproteome, proteome, and kinome. Using this integrated proteomics approach, we identified several potential kinase mediators of cabozantinib resistance and confirmed the contribution of AKT1 to erlotinib resistance in TNBC-resistant cell lines.

CSNK2 in cancer: pathophysiology and translational applications.

Protein kinase CSNK2 (CK2) is a pleiotropic serine/threonine kinase frequently dysregulated in solid and hematologic malignancies. To consolidate a wide range of biological and clinically oriented data from this unique kinase in cancer, this systematic review summarises existing knowledge from in vitro, in vivo and pre-clinical studies on CSNK2 across 24 different human cancer types. CSNK2 mRNA transcripts, protein levels and activity were found to be routinely upregulated in cancer, and commonly identified phosphotargets included AKT, STAT3, RELA, PTEN and TP53. Phenotypically, it frequently influenced evasion of apoptosis, enhancement of proliferation, cell invasion/metastasis and cell cycle control. Clinically, it held prognostic significance across 14 different cancers, and its inhibition in xenograft experiments resulted in a positive treatment response in 12. In conjunction with commentary on preliminary studies of CSNK2 inhibitors in humans, this review harmonises an extensive body of CSNK2 data in cancer and reinforces its emergence as an attractive target for cancer therapy. Continuing to investigate CSNK2 will be crucial to advancing our understanding of CSNK2 biology, and offers the promise of important new discoveries scientifically and clinically.

CK2 Regulation: Perspectives in 2021.

The protein kinase CK2 (CK2) family encompasses a small number of acidophilic serine/threonine kinases that phosphorylate substrates involved in numerous biological processes including apoptosis, cell proliferation, and the DNA damage response. CK2 has also been implicated in many human malignancies and other disorders including Alzheimer's and Parkinson's diseases, and COVID-19. Interestingly, no single mechanism describes how CK2 is regulated, including activation by external proteins or domains, phosphorylation, or dimerization. Furthermore, the kinase has an elongated activation loop that locks the kinase into an active conformation, leading CK2 to be labelled a constitutively active kinase. This presents an interesting paradox that remains unanswered: how can a constitutively active kinase regulate biological processes that require careful control? Here, we highlight a selection of studies where CK2 activity is regulated at the substrate level, and discuss them based on the regulatory mechanism. Overall, this review describes numerous biological processes where CK2 activity is regulated, highlighting how a constitutively active kinase can still control numerous cellular activities. It is also evident that more research is required to fully elucidate the mechanisms that regulate CK2 and what causes aberrant CK2 signaling in disease.

Development of a potent and selective chemical probe for the pleiotropic kinase CK2.

Building on the pyrazolopyrimidine CK2 (casein kinase 2) inhibitor scaffold, we designed a small targeted library. Through comprehensive evaluation of inhibitor selectivity, we identified inhibitor 24 (SGC-CK2-1) as a highly potent and cell-active CK2 chemical probe with exclusive selectivity for both human CK2 isoforms. Remarkably, despite years of research pointing to CK2 as a key driver in cancer, our chemical probe did not elicit a broad antiproliferative phenotype in >90% of >140 cell lines when tested in dose-response. While many publications have reported CK2 functions, CK2 biology is complex and an available high-quality chemical tool such as SGC-CK2-1 will be indispensable in deciphering the relationships between CK2 function and phenotypes.

Pannexin 1 mutation found in melanoma tumor reduces phosphorylation, glycosylation, and trafficking of the channel-forming protein.

Pannexin 1 (PANX1) is a glycoprotein that forms large pore channels capable of passing ions and metabolites such as ATP for cellular communication. PANX1 has been implicated in many diseases including breast cancer and melanoma, where inhibition or deletion of PANX1 reduced the tumorigenic and metastatic properties of the cancer cells. We interrogated the effect of single amino acid changes in various PANX1 domains using naturally occurring variants reported in cancer patient tumors. We found that a previously reported variant (Q5H) is present in cancer cells, but was not different from the wild type (Q5) in glycosylation, trafficking, or channel function and did not affect cellular properties. We discovered that the Q5H variant is in fact the highly conserved ancestral allele of PANX1 with 89% of humans carrying at least one Q5H allele. Another mutated form Y150F, found in a melanoma patient tumor, prevented phosphorylation at Y150 as well as complex N-glycosylation while increasing intracellular localization. Sarcoma (SRC) is the predicted kinase to phosphorylate the Y150 residue, and its phosphorylation is not likely to be constitutive, but rather dynamically regulated. The Y150 phosphorylation site is the first one reported to play a role in regulating posttranslational modifications and trafficking of PANX1, with potential consequences on its large-pore channel structure and function in melanoma cells.

BIRC6 mediates imatinib resistance independently of Mcl-1.

Baculoviral IAP repeat containing 6 (BIRC6) is a member of the inhibitors of apoptosis proteins (IAPs), a family of functionally and structurally related proteins that inhibit apoptosis. BIRC6 has been implicated in drug resistance in several different human cancers, however mechanisms regulating BIRC6 have not been extensively explored. Our phosphoproteomic analysis of an imatinib-resistant chronic myelogenous leukemia (CML) cell line (MYL-R) identified increased amounts of a BIRC6 peptide phosphorylated at S480, S482, and S486 compared to imatinib-sensitive CML cells (MYL). Thus we investigated the role of BIRC6 in mediating imatinib resistance and compared it to the well-characterized anti-apoptotic protein, Mcl-1. Both BIRC6 and Mcl-1 were elevated in MYL-R compared to MYL cells. Lentiviral shRNA knockdown of BIRC6 in MYL-R cells increased imatinib-stimulated caspase activation and resulted in a ~20-25-fold increase in imatinib sensitivity, without affecting Mcl-1. Treating MYL-R cells with CDK9 inhibitors decreased BIRC6 mRNA, but not BIRC6 protein levels. By contrast, while CDK9 inhibitors reduced Mcl-1 mRNA and protein, they did not affect imatinib sensitivity. Since the Src family kinase Lyn is highly expressed and active in MYL-R cells, we tested the effects of Lyn inhibition on BIRC6 and Mcl-1. RNAi-mediated knockdown or inhibition of Lyn (dasatinib/ponatinib) reduced BIRC6 protein stability and increased caspase activation. Inhibition of Lyn also increased formation of an N-terminal BIRC6 fragment in parallel with reduced amount of the BIRC6 phosphopeptide, suggesting that Lyn may regulate BIRC6 phosphorylation and stability. In summary, our data show that BIRC6 stability is dependent on Lyn, and that BIRC6 mediates imatinib sensitivity independently of Mcl-1 or CDK9. Hence, BIRC6 may be a novel target for the treatment of drug-resistant CML where Mcl-1 or CDK9 inhibitors have failed.

Cellular interaction influenced by surface modification strategies of gelatin-based nanoparticles.

Theranostic applications of gelatin nanospheres require two major components, a method of detection and good biocompatibility. We characterized the response of UTA-6 human osteosarcoma cells to the introduction of functionalized 90 bloom-based gelatin nanospheres (158 ± 49 nm) modified with three elements in different order: (a) hybridization with cadmium-based quantum dots for optical detection, (b) bioconjugation with anti-human IgG FAB (anti-IgG) for cell targeting, with/without (c) capping with polyethylene glycol on the surface for enhanced biocompatibility. A one-pot process is developed for incorporating quantum dots and antibody with gelatin nanospheres. Path A of modifying gelatin nanospheres with quantum dots first followed by anti-IgG resulted in a significantly greater cellular viability than Path B with anti-IgG first followed by quantum dots. Capping with polyethylene glycol as the final step in modification yielded significantly opposing results with decreases in Path A and increases in Path B. Three-dimensional z-stacking fluorescent images of hybrid gelatin nanospheres with anti-IgG is observed to have an increase in cellular association. The observed results suggest the modification order for building hybrid nanospheres may have an impact on cellular response.

Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.

We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified ∼20,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2 domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation. Cell-type-specific global kinase activation patterns inferred from label-free quantitation of Tyr phosphorylation guided the design of experiments to inhibit cancer cell proliferation by blocking the highly activated tyrosine kinases. Therefore, the superbinder is a highly efficient and cost-effective alternative to conventional antibodies for systematic and quantitative characterization of the tyrosine phosphoproteome under normal or pathological conditions.

Molecular Pathways: Emergence of Protein Kinase CK2 (CSNK2) as a Potential Target to Inhibit Survival and DNA Damage Response and Repair Pathways in Cancer Cells.

Protein kinase CK2 (designated CSNK2) is a constitutively active protein kinase with a vast repertoire of putative substrates that has been implicated in several human cancers, including cancer of the breast, lung, colon, and prostate, as well as hematologic malignancies. On the basis of these observations, CSNK2 has emerged as a candidate for targeted therapy, with two CSNK2 inhibitors in ongoing clinical trials. CX-4945 is a bioavailable small-molecule ATP-competitive inhibitor targeting its active site, and CIGB-300 is a cell-permeable cyclic peptide that prevents phosphorylation of the E7 protein of HPV16 by CSNK2. In preclinical models, either of these inhibitors exhibit antitumor efficacy. Furthermore, in combinations with chemotherapeutics such as cisplatin or gemcitabine, either CX-4945 or CIGB-300 promote synergistic induction of apoptosis. While CSNK2 is a regulatory participant in many processes related to cancer, its potential to modulate caspase action may be particularly pertinent to its emergence as a therapeutic target. Because the substrate recognition motifs for CSNK2 and caspases are remarkably similar, CSNK2 can block the cleavage of many caspase substrates through the phosphorylation of sites adjacent to cleavage sites. Phosphoproteomic strategies have also revealed previously underappreciated roles for CSNK2 in the phosphorylation of several key constituents of DNA damage and DNA repair pathways. Going forward, applications of proteomic strategies to interrogate responses to CSNK2 inhibitors are expected to reveal signatures for CSNK2 inhibition and molecular insights to guide new strategies to interfere with its potential to inhibit caspase action or enhance the susceptibility of cancer cells to DNA damage. Clin Cancer Res; 22(12); 2840-7. ©2016 AACR.

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly.

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2ß phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2ß mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2ß and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.

Peroxide-mediated oxidation and inhibition of the peptidyl-prolyl isomerase Pin1.

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimer's disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pK(a), we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO(2)H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO(2)H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease.

Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.

An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate phosphorylation.

Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.

Mitotic regulation of SEPT9 protein by cyclin-dependent kinase 1 (Cdk1) and Pin1 protein is important for the completion of cytokinesis.

Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.

Characterizing the convergence of protein kinase CK2 and caspase-3 reveals isoform-specific phosphorylation of caspase-3 by CK2α': implications for pathological roles of CK2 in promoting cancer cell survival.

Protein kinase CK2 has emerged as a promising candidate for the treatment of a number of cancers. This enzyme is comprised of two catalytic subunits (CK2 and/or CK2α') that form complexes with homodimers of regulatory CK2ß subunits. While catalytic and regulatory CK2 subunits are generally expressed at similar levels to form tetrameric complexes, asymmetric expression of CK2 subunits has been associated with various forms of cancer and the enhanced survival of cancer cells. To elucidate mechanisms responsible for regulation of cancer cell survival by CK2, we recently employed computational and experimental strategies that revealed widespread overlap between sites for CK2 phosphorylation and caspase cleavage. Among candidates with overlapping CK2 and caspase cleavage sites was caspase-3 that is phosphorylated by CK2 to prevent its activation by upstream caspases. To elucidate the precise relationship between CK2 and caspase-3, we modulated expression of individual CK2 subunits and demonstrated that CK2α' exhibits a striking preference for caspase-3 phosphorylation in cells as compared to CK2α and that CK2ß exhibits the capacity to abolish caspase-3 phosphorylation. Since caspase-3 represents the first CK2 substrate selectively phosphorylated by CK2α' in cells, our work highlights divergent functions of the different forms of CK2. Given the involvement of CK2 in a diverse series of biological events and its association with various cancers, this work has important implications for identifying pathological roles of distinct forms of CK2 that could instruct efforts to selectively target individual CK2 subunits for therapy.

Chemical proteomics and functional proteomics strategies for protein kinase inhibitor validation and protein kinase substrate identification: applications to protein kinase CK2.

Since protein kinases have been implicated in numerous human diseases, kinase inhibitors have emerged as promising therapeutic agents. Despite this promise, there has been a relative lag in the development of unbiased strategies to validate both inhibitor specificity and the ability to inhibit target activity within living cells. To overcome these limitations, our efforts have been focused on the development of systematic strategies that employ chemical and functional proteomics. We utilized these strategies to evaluate small molecule inhibitors of protein kinase CK2, a constitutively active kinase that has recently emerged as target for anti-cancer therapy in clinical trials. Our chemical proteomics strategies used ATP or CK2 inhibitors immobilized on sepharose beads together with mass spectrometry to capture and identify binding partners from cell extracts. These studies have verified that interactions between CK2 and its inhibitors occur in complex mixtures. However, in the case of CK2 inhibitors related to 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), our work has also revealed off-targets for the inhibitors. To complement these studies, we devised functional proteomics approaches to identify proteins that exhibit decreases in phosphorylation when cells are treated with CK2 inhibitors. To identify and validate those proteins that are direct substrates for CK2, we have also employed mutants of CK2 with decreased inhibitor sensitivity. Overall, our studies have yielded systematic platforms for studying CK2 inhibitors which we believe will foster efforts to define the biological functions of CK2 and to rigorously investigate its potential as a candidate for molecular-targeted therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Non-catalytic participation of the Pin1 peptidyl-prolyl isomerase domain in target binding.

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase (PPIase) that has the potential to add an additional level of regulation within protein kinase mediated signaling pathways. Furthermore, there is a mounting body of evidence implicating Pin1 in the emergence of pathological phenotypes in neurodegeneration and cancer through the isomerization of a wide variety of substrates at peptidyl-prolyl bonds where the residue preceding proline is a phosphorylated serine or threonine residue (i.e., pS/T-P motifs). A key step in this regulatory process is the interaction of Pin-1 with its substrates. This is a complex process since Pin1 is composed of two domains, the catalytic PPIase domain, and a type IV WW domain, both of which recognize pS/T-P motifs. The observation that the WW domain exhibits considerably higher binding affinity for pS/T-P motifs has led to predictions that the two domains may have distinct roles in mediating the actions of Pin1 on its substrates. To evaluate the participation of its individual domains in target binding, we performed GST pulldowns to monitor interactions between various forms of Pin1 and mitotic phospho-proteins that revealed two classes of Pin-1 interacting proteins, differing in their requirement for residues within the PPIase domain. From these observations, we consider models for Pin1-substrate interactions and the potential functions of the different classes of Pin1 interacting proteins. We also compare sequences that are recognized by Pin1 within its individual interaction partners to investigate the underlying basis for its different types of interactions.

Loss of pannexin 1 attenuates melanoma progression by reversion to a melanocytic phenotype.

Pannexin 1 (Panx1) is a channel-forming glycoprotein expressed in different cell types of mammalian skin. We examined the role of Panx1 in melanoma tumorigenesis and metastasis since qPCR and Western blots revealed that mouse melanocytes exhibited low levels of Panx1 while increased Panx1 expression was correlated with tumor cell aggressiveness in the isogenic melanoma cell lines (B16-F0, -F10, and -BL6). Panx1 shRNA knockdown (Panx1-KD) generated stable BL6 cell lines, with reduced dye uptake, that showed a marked increase in melanocyte-like cell characteristics including higher melanin production, decreased cell migration and enhanced formation of cellular projections. Western blotting and proteomic analyses using 2D-gel/mass spectroscopy identified vimentin and ß-catenin as two of the markers of malignant melanoma that were down-regulated in Panx1-KD cells. Xenograft Panx1-KD cells grown within the chorioallantoic membrane of avian embryos developed tumors that were significantly smaller than controls. Mouse-Alu qPCR of the excised avian embryonic organs revealed that tumor metastasis to the liver was significantly reduced upon Panx1 knockdown. These data suggest that while Panx1 is present in skin melanocytes it is up-regulated during melanoma tumor progression, and tumorigenesis can be inhibited by the knockdown of Panx1 raising the possibility that Panx1 may be a viable target for the treatment of melanoma.