Abstract ID 129650Poster Board 454Melanoma remains as the most aggressive and lethal form of skin cancer for which new therapies are still needed. Current treatment options include conventional chemotherapy, immunotherapy, and targeted therapy. Disadvantages of these therapies, as well as other factors such as refractiveness, have contributed to poor outcomes in melanoma patients. Antifungal drugs potentially represent a novel class of anticancer agents that may lead to improved therapeutic outcomes for these patients. Our objective here was to analyze the ability of imidazole antifungal agents to selectively induce cell death in primary human metastatic melanoma cells, including those harboring well-known cancer mutations.The induction of cell death was analyzed by flow cytometry and immunoblotting detection of pro-cell death proteins. Wild-type human melanoma cell lines or those possessing the BRAF V600E mutation or the NRAS Q61R mutation were analyzed. All imidazole antifungal agent treatments (clotrimazole, econazole, and miconazole) led to significant increases in cell death in wild-type, BRAF, and NRAS mutant melanoma cells as revealed by flow cytometry. Positive immunoblot detection of activated caspase-9 and cleaved poly(ADP-ribose) polymerase-1 (PARP1) by Western blot in wild-type, BRAF, and NRAS mutant melanoma cells verified the induction of caspase-dependent cell death by the antifungal agent treatments. No significant cell death or detection of pro-cell death proteins was observed in normal skin cells and untreated melanoma cells. These results indicated that clotrimazole, econazole, and miconazole were effective in selectively inducing cell death in human metastatic melanoma cell lines while leaving normal skin cells unharmed.In conclusion, this study demonstrated the ability of antifungal agents to induce cell death, an important antitumor effect, in human metastatic melanoma cells. Not only was this cell death selectively induced in melanoma cells, but it also was induced in melanoma cells that harbor well-known driver mutations in cancer. This data provides additional evidence that imidazole antifungal agents potentially represent a novel class of drugs that can successfully treat melanoma.

The transient receptor potential, the melastatin (TRPM) subfamily, which consists of eight known members, appears to have significant importance in melanoma progression, treatment, and prognosis. As several members were originally cloned from cancerous tissue, initial studies aimed towards identifying TRPM involvement in cancer progression and tumorigenesis. For relevance in skin cancer, previous research has shown roles for several TRPM members in skin cancer progression, growth, and patient prognosis. One unique member, TRPM2, appears to have notable therapeutic potential in the treatment of melanoma. Previous and recent studies have demonstrated increased TRPM2 expression levels in melanoma, as well as important roles for TRPM2 in melanoma growth, proliferation, and survival. TRPM2 is thus an emerging target in the treatment of melanoma, where TRPM2 antagonism may offer an additional treatment option for melanoma patients in the future.

Transient receptor potential melastatin-2 (TRPM2) is an emerging chemotherapeutic target due to its involvement in poly(ADP-ribose) metabolism and the ability to induce anticancer effects after antagonism of its functions. Normally functioning as a nonspecific cation channel that is activated by free ADP-ribose, TRPM2 is involved with many cellular processes, including the induction of cell death after oxidative stress. What is becoming clear is that antagonism of TRPM2 selectively induces anticancer effects in several types of cancer. We previously demonstrated decreased growth and proliferation, increased levels of DNA damage, and the selective induction of cell death in breast cancer and melanoma cells. Due to these effects, it appears that TRPM2 has a novel role in cancer cells. Further, this novel role appears to involve nuclear function, because our studies, as well as those from other independent groups, demonstrate a nuclear localization of TRPM2 in various types of cancers. Thus, as an emerging therapeutic target, it is important to describe research techniques that can be utilized to analyze TRPM2 function, determine its effects in cancerous and noncancerous cells, and provide molecular biological methods to inhibit or downregulate its function.

ID 19624 Poster Board 241 The incidence of melanoma, the most aggressive and lethal form of skin cancer, continues to rise each year in the United States. Current treatment options include conventional chemotherapy, immunotherapy, and targeted therapy. Disadvantages of these therapies, such as refractiveness and resistance, have contributed to poor outcomes in a significant number of melanoma patients. Therefore, there remains a need for new treatments. Imidazole antifungal agents potentially represent a class of new anticancer agents. Our objective here was to analyze the ability of various imidazole antifungal agents to exert anticancer effects in primary human metastatic melanoma cells, including those harboring well-known cancer mutations. The anticancer effects investigated were the induction of cell death via flow cytometry and decreased growth and proliferation via cell growth assay. Wild-type human melanoma cells, melanoma cells possessing the BRAF V600E mutation, and melanoma cells with the NRAS Q61R mutation were analyzed. The imidazole antifungal agents, clotrimazole, econazole, and miconazole, each induced significant increases in cell death and decreases in growth and proliferation in wild-type and Braf/Nras melanoma cells with doses of 10-50 μM. Of the antifungal agents utilized, overall clotrimazole resulted in the most efficacious effects. Interestingly, in Braf-mutant human melanoma cells, the antifungal agents led to profound decreases in cell growth but lesser effects on cell death. This may indicate an effect on cell cycle progression in Braf mutant cells. Taken together, these studies indicated that imidazole antifungal agents were effective in inducing antitumor effects in human metastatic melanoma cell lines, but with differing degrees of effects on cell death. This data suggests that melanoma tumors of each known genotype can potentially be treated with imidazole antifungal agents. In conclusion, this study demonstrated the ability of imidazole antifungal agents to induce anticancer effects in human metastatic melanoma cells, including those harboring well-known cancer mutations. Since differing levels of effects were observed using different antifungal agents, it is possible that each agent utilized affected different molecular targets in the melanoma cells. These antifungal agents thus appear to be potential alternative treatments, as well as affordable and widely accessible therapies, for melanoma patients in the future.

Melanoma continues to be the most aggressive and devastating form of skin cancer for which the development of novel therapies is required. The present study aimed to determine the effects of antagonism of the transient receptor potential melastatin‑2 (TRPM2) ion channel in primary human malignant melanoma cells. TRPM2 antagonism via use of the antifungal agent, clotrimazole, led to decreases in cell proliferation, as well as dose‑dependent increases in cell death in all melanoma cell lines investigated. The targeting of TRPM2 channels was verified using TRPM2 knockdown, where treatment with TRPM2 small‑interfering RNA led to similar levels of cell death in all melanoma cell lines when compared with clotrimazole treatment. Minimal effects on proliferation and cell death were observed following antagonism or knockdown of TRPM2 in non‑cancerous human keratinocytes. Moreover, characteristics of TRPM2 were explored in these melanoma cells and the results demonstrated that TRPM2, localized to the plasma membrane as a non‑specific ion channel in non‑cancerous cells, displayed a nuclear localization in all human melanoma cell lines analyzed. Additional characterization of these melanoma cell lines confirmed that each expressed one or more established multidrug resistance genes. Results of the present study therefore indicated that antagonism of the TRPM2 channel led to antitumor effects in human melanoma cells, including those that are potentially unresponsive to current treatments due to the expression of drug resistance genes. The unique cellular localization of TRPM2 and the specificity of the antitumor effects elicited by TRPM2 antagonism suggested that TRPM2 possesses a unique role in melanoma cells. Collectively, the targeting of TRPM2 represents a potentially novel, efficacious and readily accessible treatment option for patients with melanoma.

Melanoma remains as the most aggressive and lethal form of skin cancer for which new therapies are still needed. Current treatment options include conventional chemotherapy, immunotherapy, and targeted therapy. Disadvantages of these therapies, as well as other factors such as refractiveness, have contributed to poor outcomes in melanoma patients. Antifungal drugs potentially represent a novel class of anticancer agents. Our objective here was to analyze the ability of these agents to eradicate primary human metastatic melanoma cells. Antitumor effects were investigated using cell growth and proliferation assays, flow cytometry (FACS), and cell death assays. Wild-type human melanoma cell lines or those possessing the BRAF V600E mutation or the NRAS Q61R mutation were analyzed. Clotrimazole treatment led to profound decreases in cell growth in wild-type and NRAS mutant melanoma cells. Significant, but lesser effects were observed in BRAF mutants. However, treatment with econazole caused substantial reductions in cell growth in wild-type and BRAF mutant cells. These results were corroborated by cell death analyses and FACS, where clotrimazole treatment caused greater than 80% cell death in wild-type and NRAS mutant cells, while econazole caused greater than 75% cell death in BRAF mutants. Taken together, these preliminary studies indicated that clotrimazole and econazole were effective in inducing antitumor effects in human metastatic melanoma cell lines, but with differing degrees of efficacy. This data suggests that melanoma tumors of each known genotype can potentially be treated with antifungal agents. In conclusion, this study demonstrated the ability of antifungal agents to successfully treat human metastatic melanoma cells. Since each agent displayed differential effects for each type of mutation, it is possible that these antifungal agents affect different molecular targets in each melanoma cell line. These antifungal agents thus appear to be potential alternative treatments, as well as affordable and widely accessible therapies, for melanoma patients in the future. Citation Format: Lukas R. Jira, Hattie M. Foster, Hannah K. Hall, David W. Koh. Investigation of antitumor activity induced by antifungal agents in human metastatic melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4054.

Approximately 70% of all melanomas are characterized by mutations in the BRAF or NRAS genes. Although targeted therapy exists for BRAF mutations, this therapy is not commonly used in wild‐type melanoma, no such treatment is available for NRAS mutations, and resistance to all available treatments is increasing. These observations indicate that new strategies and targets are needed to improve melanoma therapy. We previously reported the potential for the antifungal agents, clotrimazole and econazole, to treat melanoma. Our objective here was to analyze the ability of these agents to treat human melanoma cell lines expressing the BRAF or NRAS mutations. Cell growth and cell death were investigated via flow cytometry, cell proliferation assays, and cell death assays after treatments.Wild‐type human melanoma cell lines or those possessing the BRAF V600E mutation or the NRAS Q61R mutation were analyzed. A dose of 25 μM clotrimazole led to profound decreases in cell growth in wild‐type and NRAS mutant melanoma cells. Significant, but lesser effects were observed in BRAF mutants. However, treatment with 25 μM econazole caused substantial reductions in cell growth in BRAF mutant cells, as well as one line of wild‐type melanoma cells. These results were corroborated by cell death analyses, where clotrimazole treatment caused up to 50% cell death in wild‐type and NRAS mutant cells after 3 days. In BRAF mutants after 3 days, econazole treatment caused up to 45% cell death. Taken together, these preliminary studies indicated that clotrimazole was more effective in the treatment of wild‐type and NRAS mutant melanoma cells, while econazole demonstrated greater effects in melanoma cells expressing the BRAF mutation.In conclusion, this study demonstrated the ability of antifungal agents to successfully treat human melanoma cell lines that possess BRAF and NRAS mutations. Since each agent displayed differential effects for each type of mutation, it is possible that these antifungal agents affect different molecular targets in each mutant melanoma cell line. These antifungal agents thus appear to be potential alternative treatments for wild‐type melanoma or melanoma characterized with the BRAF V600E or NRAS Q61R mutation.

Patients with melanoma, the most aggressive form of skin cancer, have poor survival rates due to the resistance of advanced stage tumors to chemotherapy. This resistance appears to involve the dysregulation of apoptosis, such that inhibition of apoptotic progression allows melanoma cells to survive after treatment. We previously reported a potentially novel treatment for melanoma, where antifungal agents led to profound decreases in growth and proliferation of human melanoma cells. Our objective here was to analyze cell death pathways induced in human melanoma cell lines following treatment with antifungal agents. Cell death pathways were investigated via flow cytometry, cell proliferation assays, and immunoblotting after treatments.Several lines of human melanoma and normal skin cells were analyzed. Apoptosis, via flow cytometry and analysis of activated caspases and PARP1 cleavage, was observed in all melanoma lines after treatment with clotrimazole and econazole. Caspase‐independent cell death was also observed in most melanoma lines after similar treatments. No extensive cell death was induced by these treatments in noncancerous human skin cells. These results indicated that antifungal agents selectively induced cytotoxicity in human melanoma lines. Because varying levels of cell death were observed among different melanoma lines after antifungal treatment, further studies will be required to ascertain the reason for these differential responses to treatment. Taken together, these results indicated that clotrimazole and econazole initiated apoptotic and non‐apoptotic cell death in human melanoma cells.In conclusion, this study demonstrated the induction of apoptotic and non‐apoptotic cell death in human melanoma skin cell lines after treatment with antifungal agents. Differential levels of various cell death pathways were observed among all melanoma lines. This was corroborated by the various levels of efficacious treatment produced by these agents. Taken together, we conclude that clotrimazole and econazole induce cell death in human melanoma cells. These agents thus appear to have in vitro efficacy toward the treatment of human melanoma.Support or Funding InformationThis research was supported in part by the Bower, Bennet & Bennet Endowed Chair Award from the Ohio Northern University College of Pharmacy and the Ohio Northern University Summer Research stipend.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The multidrug resistance (MDR) phenotype allows cancer cells to display decreased sensitivity to chemotherapy. A well‐characterized drug resistance gene in cancer is MDR1, which encodes for the p‐glycoprotein/ATP‐binding cassette (ABC) transporter. Lesser known are the roles of the multidrug resistance‐associated protein 1 (MRP1) and lung resistance‐related protein (LRP) in melanoma. Because of the previously demonstrated presence of MRP1 and LRP in human melanoma cells and the continued difficulty of overcoming MDR in melanoma treatment, here we characterized the expression levels of drug resistance genes in melanoma cell lines derived from human patients and determined their susceptibility to novel antifungal treatment. Expression levels were determined by real‐time PCR (qPCR) and immunoblot analyses. Susceptibility of cell lines to antifungal agents was analyzed by flow cytometry, cell proliferation assays, and immunoblotting after drug treatments.Several lines of human melanoma and normal skin cells were analyzed. MDR1 expression was observed in multiple melanoma lines, while increased levels of MRP1 were observed in normal skin cells and one melanoma line. Interestingly, LRP expression was highest in normal skin cells and several melanoma lines. Novel treatment with clotrimazole, an agent previously shown to eradicate melanoma cells, caused significant decreases in proliferation and increases in cell death in all MDR melanoma lines. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cells. These results indicated that clotrimazole selectively induces cytotoxicity in drug‐resistant melanoma lines. However, decreased efficacy was observed in melanoma lines that overexpressed LRP, as compared to all other melanoma lines that overexpressed MDR1 and/or MRP1. These results indicated that LRP, mostly known for facilitating drug resistance in lung and breast cancer, may also have a significant role in the ability of melanoma cells to resist the effects of conventional melanoma treatments.In conclusion, this study demonstrated the existence and differential expression levels of MDR1, MRP1, and LRP in melanoma and normal skin cell lines. Although all melanoma lines were susceptible to treatment with clotrimazole, decreased efficacy was observed in melanoma lines that overexpressed LRP. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug‐resistant human melanoma cells. However, these preliminary studies also indicated that LRP is potentially a significant facilitator of drug resistance in human melanoma.Support or Funding InformationThis research was funded by the Ohio Northern University Summer Research Stipend.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A recent article published in Scientific Reports presents compelling evidence that a member of the transient receptor potential melastatin (TRPM) family of plasma membrane ion channels has essential roles in the survival and migration of tongue carcinoma cells. The work thus identifies the targeting of this ion channel, TRPM2, as a potential strategy to selectively treat oral cancer. Previously, TRPM2 was reported to have similar roles in other types of cancer, such as breast cancer, metastatic melanoma, prostate cancer, and neuroblastoma. Accordingly, this study offers further evidence that multiple types of cancer appear to utilize TRPM2 in a unique fashion, most likely in order to survive and proliferate. Intriguingly, this alternative use of TRPM2 appears to occur in the nuclei of these cancer cells. Elucidating this nuclear role and further determining the extent of this nuclear function of TRPM2 in other cancers may provide the data necessary to selectively and efficaciously treat human cancers in the future.

Abstract Drug resistance to chemotherapy, mediated in part by overexpression of P-glycoprotein (MDR-1), multidrug resistance-associated protein (MRP), or lung resistance-realted protein (LRP), continues to be a major problem in the treatment of melanoma. Because we previously demonstrated the ability of clotrimazole to enhance cytotoxicity in human metastatic melanoma cells, here we evaluated the ability of clotrimazole to treat drug-resistant human melanoma cells that overexpress MRP and/or LRP. We utilized human melanoma cell lines that overexpress either MDR or LRP, and human melanoma cell lines that overexpress both. Cell growth and cell death were analyzed by flow cytometry, proliferation assays, and immunoblotting after drug treatments and RNA interference. Clotrimazole caused significant decreases in proliferation and increases in cell death in both drug-sensitive melanoma lines and those overexpressing MRP and LRP. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cell lines. These results indicate that clotrimazole selectively induces cytotoxicity in both drug-sensitive and drug-resistant melanoma lines. As clotrimazole is known to inhibit various transient receptor potential (TRP) ion channels, we pretreated these melanoma lines with TRP inhibitors or RNAi. Inhibitors of the TRP melastatin-8 channel (TRPM8) or TRP vanilloid-1 (TRPV1) failed to produce comparable levels of cell death caused by clotrimazole. However, RNAi silencing of the TRPM2 cation channel caused significant levels of cell death in both drug-sensitive and drug-resistant melanoma lines. These results indicated that inhibition of TRPM2 channels may have a primary role in the ability of clotrimazole to treat drug-sensitive and drug-resistant melanoma lines. In conclusion, this study demonstrated that clotrimazole selectively increases cell death in drug-resistant melanoma lines, with minimal deleterious effects in normal skin cells. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug-resistant human melanoma cells. Thus, these preliminary studies indicate that clotrimazole has the potential to successfully treat drug resistant melanoma in the future. Citation Format: Steven D. Blake, Shelby G. McKamey, Christopher M. Tweed, David W. Koh. Evaluation of the efficacy of clotrimazole treatment in human melanoma cell lines that overexpress the multidrug resistance-associated protein (MRP) and the lung resistance-related protein (LRP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2017-16

Abstract The transient receptor potential melastatin-2 (TRPM2) cation channel was previously identified as a potential target in various cancers, where its pharmacologic inhibition caused increased DNA damage and the selective eradication of cancer cells. In this study, we utilized several human primary metastatic melanoma cell lines, in which we analyzed TRPM2 and its isoforms, and evaluated the ability of TRPM2 inhibition to modulate cell death. As TRPM2 is known to exist as a plasma membrane cation channel in noncancerous cells, we investigated the localization of TRPM2 in melanoma cells. In three lines of primary human metastatic melanoma cells, TRPM2 was localized in the nucleus, as compared to an extra-nuclear localization in noncancerous primary epidermal keratinocyes. Because TRPM2 exists in several isoforms in cancer cells, we investigated the cellular localization of these isoforms. Subcellular fractionations demonstrated that only full-length TRPM2 was localized to the nucleus in primary human metastatic melanoma cells, whereas two smaller isoforms were localized exclusively in the cytoplasmic fraction. Treatment with the TRPM2 inhibitor, clotrimazole, caused decreased proliferation in all three lines of primary human metastatic melanoma cells. Further, treatment with the DNA alkylating agent, temozolomide (TMZ), led to increased levels of cell death in melanoma cells pretreated with clotrimazole, but not in noncancerous primary epidermal keratinocyes after pretreatment. Similar increases in cell death were observed after RNAi silencing of TRPM2 followed by TMZ treatment. Taken together, this study demonstrated that TRPM2 inhibition selectively increases cell death in primary human metastatic melanoma cells. Further, the data suggests that, similar to our previous results in breast adenocarcinoma cells, full-length TRPM2 appears to have a novel role in melanoma cell growth and survival. The results therefore suggest that TRPM2 is a potential target in melanoma, where its inhibition may cause the selective eradication of metastatic melanoma. Citation Format: David W. Koh, Steven D. Blake, Daniel P. Powell. Enhanced cytotoxicity in primary human metastatic melanoma cells via inhibition of the transient receptor potential melastatin-2 (TRPM2) channel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1269.

Transient receptor potential, melastatin-2 (TRPM2) is a plasma membrane cation channel with important roles in sensory functions and promoting cell death. However, we demonstrated here that TRPM2 was present in the nuclei of MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, and its pharmacologic inhibition or RNAi silencing caused decreased cell proliferation. Neither an effect on proliferation nor a localization of TRPM2 in the nucleus was observed in noncancerous HMEC and MCF-10A human mammary epithelial cells. Investigation of possible effects of TRPM2 function in the nucleus demonstrated that pharmacologic inhibition or RNAi silencing of TRPM2 in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells caused up to 4-fold increases in DNA damage levels, as compared to noncancerous breast cells after equivalent treatments. These results indicate that TRPM2 has a novel nuclear function in human breast adenocarcinoma cells that facilitates the integrity of genomic DNA, a finding that is distinct from its previously reported role as a plasma membrane cation channel in noncancerous cells. In summary, we report here a novel effect promoted by TRPM2, where it functions to minimize DNA damage and thus may have a role in the protection of genomic DNA in breast cancer cells. Our study therefore provides compelling evidence that TRPM2 has a unique role in breast adenocarcinoma cells. Accordingly, these studies suggest that TRPM2 is a potential therapeutic target, where its pharmacologic inhibition may provide an innovative strategy to selectively increase DNA damage levels in breast cancer cells.

We previously demonstrated a unique protective role for the transient receptor potential, melastatin-2 (TRPM2) cation channel in breast cancer cells. In the present study, we investigated the chemotherapeutic effects elicited by inhibiting this protective role in metastatic breast adenocarcinoma cells. TRPM2 inhibition led to dose-dependent increases in MDA-MB-231 breast adenocarcinoma cell death after treatment with doxorubicin or the DNA-methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine. Similar results were observed after RNAi silencing of TRPM2 in these cells after doxorubicin treatment. However, TRPM2 RNAi silencing also led to increased MCF-7 breast adenocarcinoma cell death after tamoxifen treatment, yet not in non-cancerous human mammary epithelial cells. These results thus revealed that TRPM2 inhibition selectively increased cytotoxicity in a triple-negative and an estrogen receptor-positive breast cancer cell line, with minimal deleterious effects in non-cancerous breast cells. Analysis of DNA damage revealed enhanced DNA damage levels in MCF-7 cells treated with doxorubicin due to TRPM2 inhibition. Analysis of cell death demonstrated that inhibition of apoptosis, caspase-independent cell death or autophagy failed to significantly reduce cell death induced by TRPM2 inhibition and chemotherapy. These results indicate that TRPM2 inhibition activates alternative pathways of cell death in breast cancer cells. Taken together, our results provide significant evidence that TRPM2 inhibition is a potential strategy to induce triple-negative and estrogen receptor-positive breast adenocarcinoma cell death via alternative cell death pathways. This is expected to provide a basis for inhibiting TRPM2 for the improved treatment of breast cancer, which potentially includes treating breast tumors that are resistant to chemotherapy due to their evasion of apoptosis.

Abstract Transient receptor potential melastatin-2 (TRPM2) is a plasma membrane cation channel with important physiologic roles in noncancerous cells. However, in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, we previously demonstrated that TRPM2 was present in the nucleus, and pharmacologic inhibition or RNAi silencing of TRPM2 led to decreased proliferation. In this study, we evaluated the ability of TRPM2 inhibition to produce cell death in normal breast epithelial cells and breast adenocarcinoma cells. Further, we analyzed the cell death pathways induced. Treatment with the DNA alkylating agent, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), or doxorubicin (Dox) led to increased levels of cell death in MDA-MB-231 breast adenocarcinoma cells pretreated with TRPM2 inhibitors, but not in noncancerous mammary epithelial cells after pretreatment. Similar increases in cell death were observed after RNAi silencing of TRPM2 in both MDA-MB-231 and MCF-7 breast adenocarcinoma cells after Dox or tamoxifen treatment, respectively. Investigation of apoptosis after TRPM2 inhibition demonstrated decreased levels of caspase activity and no significant effect of Q-VD-OPh, a pan-caspase inhibitor, on cell death induced by TRPM2 inhibition. Further, pretreatment with 3-methyl adenine, an inhibitor of autophagy, also produced no significant effect on cell death induced by TRPM2 inhibition. These studies indicate that apoptosis or autophagy are not the primary cell death pathways induced by TRPM2 inhibition in breast cancer cells. Also, RNAi silencing of poly(ADP-ribose) glycohydrolase (PARG) and apoptosis-inducing factor (AIF), two proteins involved in initiating or facilitating alternative pathways of cell death, produced minimal changes in cell death induced by TRPM2 inhibition. Taken together, this study demonstrated that TRPM2 inhibition selectively increases cell death in breast adenocarcinoma cells. Further, the data suggests that apoptosis, autophagy, and PARG/AIF-mediated cell death are not the primarily cell death pathways involved in the cytotoxicity caused by TRPM2 inhibition. The results therefore suggest that TRPM2 is a potential target in breast cancer, where its inhibition may cause the selective eradication of breast adenocarcinoma cells. Citation Format: David W. Koh, Daniel P. Powell, Steven D. Blake, Joy L. Hoffman, Xiaoxing Feng. Selective induction of breast adenocarcinoma cell death via inhibition of the transient receptor potential melastatin-2 (TRPM2) cation channel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2015-1717

The transient receptor potential, melastatin‐2 (TRPM2) cation channel is known for facilitating cell death and is thus targeted for pharmacologic inhibition in debilitating diseases/conditions to decrease tissue injury and prevent cell death. The objective of this study was to determine the role of TRPM2 in breast cancer cells. TRPM2 function in MCF‐7 and MDA‐MB‐231 breast adenocarcinoma cells was investigated using TRPM2 pharmacologic inhibitors or RNAi silencing. In these cells, inhibition or knockdown of TRPM2 led to decreased proliferation and increased cell death after chemotherapeutic treatments. These effects were not observed after equivalent treatments in noncancerous breast cells. Cellular localization studies confirmed the plasma membrane/cytoplasmic localization of the TRPM2 ion channel in noncancerous breast cells. However, it exhibited a unique nuclear localization in breast adenocarcinoma cells. Investigation into a possible nuclear role for TRPM2 demonstrated that inhibition or RNAi silencing of TRPM2 in breast adenocarcinoma cells led to increased DNA damage and decreased genomic integrity as measured by Comet assay. Minimal effects on genomic integrity were observed in noncancerous breast cells after equivalent treatments. This data indicates that TRPM2 facilitates genomic integrity in breast adenocarcinoma cells. Taken together, these results demonstrate that TRPM2 has a novel role in facilitating breast cancer cell genomic integrity, survival, and proliferation. Because these effects are in direct contrast to the pro‐cell death role of TRPM2 in all other tissues and noncancerous cells studied, TRPM2 may represent a novel target in breast cancer treatment. More specifically, TRPM2 pharmacologic inhibition is expected to lead to the selective eradication of breast tumors. These results are therefore expected to provide a basis for inhibiting TRPM2 for the improved treatment of breast cancer patients.

Abstract Cancer health disparities represent a major public health concern in the US. Even when factors such as socioeconomic status, carcinogen exposure, and access to care are accounted for, disparities persist in the form of higher overall incidence rates and worse clinical outcomes for minority populations than for overall population. A case of point is the racial disparities in liver cancer-related mortality. Hepatitis C virus (HCV) is the most significant contributing factor in the development of hepatocellular carcinoma (HCC). In the US, African Americans (AAs) have twice the prevalence of HCV/genotype 1 infection, and develop HCC at more than twice the rate as Caucasian (CA) counterparts. African Americans are, however, less likely to respond to interferon-based therapy than CAs, and have considerably lower likelihood of receiving liver transplantation. While it is evident that viral infection with HCV is associated with the development of HCC, there are critical gaps in our understanding of the biological basis for this racial disparity. The aim of the current study was to define the molecular signatures of HCV disease progression in liver & tumor tissue samples obtained from AA & CA patients using 8-plex iTRAQ-based proteomics coupled with bioinformatics data analyses. The raw data were analyzed by the ProteinPilot v3.0 using the paragon algorithm. Searches were performed against a comprehensive database generated from SwissPort, Refseq, and Tremble protein sequences. The data were normalized for loading error & background correction. The proteins with confidence score > 90% and with at least 1 peptide of 95% identification confidence were used for further quality control & differential expression analysis. The quality control analysis was performed using pairwise correlation plots, boxplots, principal component analysis (PCA), and unsupervised hierarchical clustering. Supervised analysis was performed to identify differentially expressed proteins (DEP), where the relative protein expression values were compared between groups (Normal vs. Cirrhosis (CIR), Normal vs. HCC, CIR vs. HCC). Based on our experimental design, 787 unique proteins were identified. Of those, 32 were differentially expressed between normal, cirrhosis & HCC groups. Targets validation using real-time PCR (RT-PCR) or western blotting (WB) shows racially distinct alteration in the expression of certain targets. For example, the mRNA expression levels of TF were 2 and18-fold higher in CIR & HCC, respectively in AAs compared to CAs. Similarly, the expression of APOA1 mRNA levels was 7-fold higher in HCC of AAs compared to CAs. This trend was similar to their protein expression levels using WB. However, the level of HNF4α protein was down regulated in AAs compared to CAs. This indicates that HNF4α does not regulate TF & APOA1 expression in HCC of AA samples. Citation Format: Simon T. Dillon, Manoj K. Bhasin, Xiaoxing (Stella) Feng, David Koh, Sayed Salih Daoud. Biomarkers discovery and racial disparity in hepatitis C-associated hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1150. doi:10.1158/1538-7445.AM2013-1150

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme that catalyzes the hydrolysis of poly(ADP-ribose) (PAR), an essential biopolymer that is synthesized by poly(ADP-ribose) polymerases (PARPs) in the cell. By regulating the hydrolytic arm of poly(ADP-ribosyl)ation, PARG participates in a number of biological processes, including the repair of DNA damage, chromatin dynamics, transcriptional regulation, and cell death. Collectively, the research investigating the roles of PARG in the cell has identified the importance of PARG and its value as a therapeutic target. However, the biological role of PARG remains less understood than the role of PAR synthesis by the PARPs. Further complicating the study of PARG is the existence of multiple PARG isoforms in the cell, the lack of optimal PARG inhibitors, and the lack of viable PARG-null animals. This review will present our current knowledge of PARG, with a focus on its roles in DNA-damage repair and cell death.

The incidence and mortality of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) is higher in African Americans (AA) than other racial/ethnic groups in the U.S., but the reasons for this disparity are unknown. There is an urgent need for the discovery of novel molecular signatures for HCV disease progression to understand the underlying biological basis for this cancer rate disparity to improve the clinical outcome.

N2O has been known to possess an analgesic property since the late 18th century. Since then, researchers have proposed different hypotheses for the mechanism, including an opioid/nitric oxide (NO) hypothesis (Emmanouil and Quock, Anesth Prog 54:9–18, 2007). Previously, we reported that N2O antinociception can be reduced by blocking NO production in the brain with selective nNOS inhibitor SMTC (Ishikawa and Quock, JPET 306:484–489, 2003). Recently, we found that increasing nNOS expression in brain contributes to HBO2 antinociception (Zhang et al., SfN Abstracts 38:575.29, 2012). The aim of this study was to discover whether N2O exposure also changes nNOS expression in the brain. Male NIH Swiss mice were exposed to 50% N2O for 60 min and sacrificed at intervals 0–8 hrs later. Brains were removed and divided into six coronal sections using a brain matrix. Expression of nNOS was examined by the western blotting assay. Results demonstrated that N2O treatment increased nNOS expression mostly in section 5 (−4.65 mm to −6.75 mm AP from bregma) and section 6 (−6.75 mm to −8.75 mm AP). The increase lasted for at least 8 hrs, which differs from HBO2‐induced nNOS expression that peaked at 6 hr. Based on these findings, we conclude that N2O exposure increased nNOS expression in the caudal mouse brain. (This research was supported by NIH Grant AT‐007222 and the Allen I. White Distinguished Professorship at Washington State University.)