Salina M. Torres

A new understanding in the epidemiology of melanoma.

The incidence of melanoma is continuing to increase worldwide. UV exposure is a known risk factor for melanoma. Geographic location is known to influence UV exposure and the distribution of the incidence of melanoma. Furthermore, epidemiologic data suggest that gender and genetics may influence the distribution of melanoma on the body surface and histopathologic characteristics of the lesion. This article describes what is known about the impact of gender, ethnicity and geography on the progression of melanoma. Advanced-stage cutaneous melanoma has a median survival time of less than 1 year. Surgical removal, radiotherapy, chemotherapy, targeted therapies and a variety of immunotherapies have been utilized in the treatment of melanoma. Current treatment strategies and the results of recent clinical trials are also discussed in this article.

WR1065 mitigates AZT‐ddI‐induced mutagenesis and inhibits viral replication

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV‐1 infection and reducing mother‐to‐child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long‐term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI‐drug pair zidovudine (AZT)–didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI‐drug pair. In TK6 cells exposed to 100 μM AZT‐ddI (equimolar) for 3 days with or without 150 μM WR1065, WR1065 enhanced long‐term cell survival and significantly reduced AZT‐ddI‐induced mutations. Follow‐up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T‐cell blasts infected with HIV‐1 in culture, inhibition of p24 protein production was observed in cells treated with 10 μM AZT in the absence or presence of 5–1,000 μM WR1065. Surprisingly, WR1065 alone exhibited dose‐related inhibition of HIV‐1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation. Environ. Mol. Mutagen. 2009.

Variants in autophagy-related genes and clinical characteristics in melanoma: a population-based study

Autophagy has been linked with melanoma risk and survival, but no polymorphisms in autophagy‐related (ATG) genes have been investigated in relation to melanoma progression. We examined five single‐nucleotide polymorphisms (SNPs) in three ATG genes (ATG5; ATG10; and ATG16L) with known or suspected impact on autophagic flux in an international population‐based case–control study of melanoma. DNA from 911 melanoma patients was genotyped. An association was identified between (GG) (rs2241880) and earlier stage at diagnosis (OR 0.47; 95% Confidence Intervals (CI) = 0.27–0.81, P = 0.02) and a decrease in Breslow thickness (P = 0.03). The ATG16L heterozygous genotype (AG) (rs2241880) was associated with younger age at diagnosis (P = 0.02). Two SNPs in ATG5 were found to be associated with increased stage (rs2245214 CG, OR 1.47; 95% CI = 1.11–1.94, P = 0.03; rs510432 CC, OR 1.84; 95% CI = 1.12–3.02, P = 0.05). Finally, we identified inverse associations between ATG5 (GG rs2245214) and melanomas on the scalp or neck (OR 0.20, 95% CI = 0.05–0.86, P = 0.03); ATG10 (CC) (rs1864182) and brisk tumor infiltrating lymphocytes (TILs) (OR 0.42; 95% CI = 0.21–0.88, P = 0.02), and ATG5 (CC) (rs510432) with nonbrisk TILs (OR 0.55; 95% CI = 0.34–0.87, P = 0.01). Our data suggest that ATG SNPs might be differentially associated with specific host and tumor characteristics including age at diagnosis, TILs, and stage. These associations may be critical to understanding the role of autophagy in cancer, and further investigation will help characterize the contribution of these variants to melanoma progression.

DNA repair variants, indoor tanning, and risk of melanoma

Although ultraviolet radiation (UV) exposure from indoor tanning has been linked to an increased risk of melanoma, the role of DNA repair genes in this process is unknown. We evaluated the association of 92 single nucleotide polymorphisms (SNPs) in 20 DNA repair genes with the risk of melanoma and indoor tanning among 929 patients with melanoma and 817 controls from the Minnesota Skin Health Study. Significant associations with melanoma risk were identified for SNPs in ERCC4, ERCC6, RFC1, XPC, MGMT, and FBRSL1 genes; with a cutoff of P < 0.05. ERCC6 and FBRSL1 gene variants and haplotypes interacted with indoor tanning. However, none of the 92 SNPs tested met the correction criteria for multiple comparisons. This study, based on an a priori interest in investigating the role of DNA repair capacity using variants in base excision and nucleotide excision repair, identified several genes that may play a role in resolving UV‐induced DNA damage.

Pan-erbB inhibition potentiates BRAF inhibitors for melanoma treatment

The BRAF inhibitor vemurafenib is currently used for treating patients with BRAF V600E mutant melanoma. However, the responses to vemurafenib are generally partial and of relatively short duration. Recent evidence suggests that activation of the epidermal growth factor receptor (EGFR)/erbB signaling pathway may be responsible for the development of BRAF inhibitor resistance in melanoma patients. In this study, we characterized the erbB family of receptors and ligands in melanoma cell lines and examined whether targeting both BRAF and erbB provided enhanced antitumor activity in BRAF mutant melanoma. Variable levels of erbB2, erbB3, and truncated erbB4 were expressed in both BRAF wildtype and mutant melanoma cells with no significant differences between wildtype and mutant lines. EGFR was rarely expressed. Neuregulin 3 and neuregulin 4 were the major erbB ligands released by melanoma cells. Multi-erbB targeting with the irreversible tyrosine kinase inhibitor canertinib exerted a more effective growth inhibitory effect in both BRAF wildtype and mutant melanoma cells compared with the single-erbB or dual-erbB targeting inhibitors, gefitinib, erlotinib, and lapatinib. Canertinib inhibited both EGF-induced and neuregulin 1-induced erbB downstream signaling in both mutant and wildtype cell lines. However, canertinib induced apoptosis and sub-G1 arrest only in mutant cells. Canertinib statistically increased the antiproliferative effects of vemurafenib in the BRAF mutant melanoma cell lines while little or no enhanced effect was observed with the combination treatment in the wildtype cell lines. A combined inhibition strategy targeting BRAF together with multiple erbB family kinases is potentially beneficial for treating BRAF V600E mutant melanoma. Wildtype BRAF melanoma may also benefit from a multi-erbB kinase inhibitor.

Abstract 3864: Biological basis of pan-ErbB receptor targeting for malignant melanoma treatment

Dysregulated activation of the epidermal growth factor receptor (ErbB) family (EGFR, ErBB2, ErbB3, and ErbB4) is involved in promoting the cellular growth of human melanoma. EGFR, ErbB3, and ErbB4 are promising targets of malignant melanoma for new molecular drug development. However, the biology of ErbB family signaling pathways indicates that functional redundancy and heterodimerization among ErbB family members can greatly compromise the therapeutic efficacy of single ErbB targeting tyrosine kinase inhibitors (TKIs). In fact, Phase II clinical trials have already shown that EGFR (ErbB1) targeting TKIs provide limited benefits to melanoma patients. Here, this study has compared single vs. multiple ErbB targeting TKIs for inhibiting the growth of melanoma cells with different ErbB family protein expression profiles. We examined the expression of ErbB family members in 14 different human melanoma cell lines (MEL-526, M14, UPCI-MEL 527.1 and 11 SK-MEL cells). EGFR was expressed in 5 of the cell lines examined (35.7 %). ErbB2 was present in 13 lines (92.9 %); ErbB3 was found in 12 lines (85.7 %); and ErbB4 was found in all the cell lines. Furthermore, individual cell lines had a particular dominant form of ErbB protein. This differential expression pattern of ErbB family proteins was also seen in melanoma tissues from patients. Out of 14 melanoma cases examined by immunohistochemistry, 8 samples (61.5 %) were positive for ErbB1 and ErbB4 expression while ErbB2 was detected in 11 samples (84.6 %). We also found that Neuregulin 3 (which ranged from 500 to 76500 pg/ml) and Neuregulin 4 (which ranged from 26-300 pg/ml), which activate ErbB3 and/or ErbB4, are the major ErbB ligands released by the human melanoma cell lines. TGFβ and Amphiregulin, which mainly bind to EGFR, were released in relatively lower levels by the majority of the cell lines, but high levels of these ligands were distinctly found in individual cell lines. Among all the cell lines examined, HB-EGF was barely undetectable. In addition, different sensitivities towards single ErbB targeting tyrosine kinase inhibitors (TKIs) and pan-ErbB targeting TKIs were seen among the melanoma cells. For example, the TKI sensitivities of MEL526 which expressed no EGFR but high ErbB2, 3, and 4 proteins, was found to be: lapatinib (IC50: 6.7 μM) > canertinib (IC50: 12.4 μM) = erlotinib (IC50: 12.9 μM) > gefitinib (IC50: 23 μM). Similarly, M14 and UPCI-MEL527.1, which also have no EGFR expression but express ErbB 2, 3, 4, also responded better to lapatinib and canertinib respectively than to gefitinib or erlotinib. Our study is the first to suggest that the pan-ErbB targeting approach may be more effective in treatment malignant melanoma. Our findings also suggest that future screening of melanoma patients for their dominantly expressed ErbB protein types can potentially serve as a guide for the choice of effective ErbB TKI in treating malignant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3864. doi:1538-7445.AM2012-3864

Quantitative Analysis of Immunohistochemistry in Melanoma Tumors

Abstract Identification of positive staining is often qualitative and subjective. This is particularly troublesome in pigmented melanoma lesions, because melanin is difficult to distinguish from the brown stain resulting from immunohistochemistry (IHC) using horse radish peroxidase developed with 3,3′-Diaminobenzidine (HRP-DAB). We sought to identify and quantify positive staining, particularly in melanoma lesions. We visualized G-protein coupled estrogen receptor (GPER) expression developed with HRP-DAB and counterstained with Azure B (stains melanin) in melanoma tissue sections (n = 3). Matched sections (n = 3), along with 22 unmatched sections, were stained only with Azure B as a control. Breast tissue (n = 1) was used as a positive HRP-DAB control. Images of the stained tissues were generated using a Nuance Spectral Imaging Camera. Analysis of the images was performed using the Nuance Spectral Imaging software and SlideBook. Data was analyzed using a Kruskal–Wallis one way analysis of variance (ANOVA). We showed that a pigmented melanoma tissue doubly stained with anti-GPER HRP-DAB and Azure B can be unmixed using spectra derived from a matched, Azure B-only section, and an anti-GPER HRP-DAB control. We unmixed each of the melanoma lesions using each of the Azure B spectra, evaluated the mean intensity of positive staining, and examined the distribution of the mean intensities (P = .73; Kruskal–Wallis). These results suggest that this method does not require a matched Azure B-only stained control tissue for every melanoma lesion, allowing precious tissues to be conserved for other studies. Importantly, this quantification method reduces the subjectivity of protein expression analysis, and provides a valuable tool for accurate evaluation, particularly for pigmented tissues.

Abstract 1016: Variants in autophagy related genes and clinical characteristics in melanoma: a population-based study

Autophagy has been linked with melanoma, but no polymorphisms in autophagy related (ATG) genes have been investigated for association with melanoma prognostic indicators and survival. We examined 5 ATG gene single nucleotide polymorphisms (SNPs) in a large international multicenter population-based case-control study of melanoma. DNA from 911 melanoma patients was genotyped for five SNPs with a known or suspected impact on autophagic flux. While we did not identify an association with survival, a significant association was identified between the minor allele for an ATG16L polymorphism (rs2241880) and a decrease in Breslow thickness (p = 0.03), earlier tumor stage at diagnosis (OR 0.47, 95% CI 0.27-0.81, p = 0.02) and younger age at diagnosis (p = 0.02). In addition, two SNPs in ATG5 (rs2245214 and rs510432) were found to be significantly associated with increased tumor stage of melanoma (OR 1.84 95% CI 1.12-3.02, p = 0.05; OR 1.47 95% CI 1.11-1.94, p = 0.03). Finally, we identified inverse associations between the minor allele of rs2245214 and melanomas on the scalp or neck (OR 0.20, 95% CI 0.05-0.86, p = 0.03); rs1864182 (OR 0.42, 95% CI 0.21-0.88, p = 0.02) and brisk TILs, and rs510432 (OR 0.55 95% CI 0.34-0.87, p = 0.01) with non-brisk TILs, although they were not globally significant. In summary, our data suggests that ATG SNPs, while not associated with survival, may be associated with Breslow thickness, tumor stage, age at diagnosis, and aggressive histopathological factors. These associations could contribute to our current understanding of the significant role of autophagy in melanoma progression. Citation Format: Kirsten A. m. White, Li Luo, Todd A. Thompson, Salina Torres, Chien-An A. Hu, Nancy E. Thomas, Hoda Anton-Culver, Stephen B. Gruber, Lynn From, Klaus J. Busam, Irene Orlow, Peter A. Kanetsky, Lorraine D. Marrett, Richard P. Gallagher, Roberto Zanetti, Stefano Rosso, Terry Dwyer, Anne E. Cust, Allison Venn, Colin B. Begg, Marianne Berwick, Jenna Lillyquist. Variants in autophagy related genes and clinical characteristics in melanoma: a population-based study. [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 1016.

Abstract 2909: LC3 protein expression associates with UV exposure in melanoma histopathology

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The 5-year survival rate for stage IV melanoma is estimated to remain unchanged at only 15%, even with new therapies. One emerging hallmark of cancer is the reprogramming of energy metabolism including the role of autophagy. In cancer, autophagy appears to be an important energy source for nutrient-depleted tumors. Ultraviolet-radiation (UVR) exposure is an established risk factor for melanoma and increases autophagic flux. We hypothesize that UV exposure is independently associated with both levels of autophagy and melanoma survival. To address this hypothesis, we are investigating the association between UVR exposure, autophagy, and development of melanoma. Basal levels of autophagy appear to vary by tumor stage, and higher levels of autophagy are associated with poor clinical prognosis. Melanoma cells contain high levels of autophagosomes, a cellular structure associated with autophagy, as measured by the proxy marker LC3. Also, the expression of the autophagy marker Beclin 1 has been associated with increased risk of early death from melanoma. These findings suggest that autophagy provides metabolic support for tumors as it localizes to hypoxic tumor areas, supports tumor cell survival, and is activated in response to stress. Autophagy can act as a cytoprotective mechanism against UV-induced apoptosis. We have shown that rates of autophagy vary by oncogene status. Autophagy was evaluated in formalin-fixed, paraffin embedded melanoma and normal tissue sections, by staining for LC3 and Beclin 1 using IHC. These markers of autophagy, along with clinical tumor characteristics, were correlated with extensive questionnaire data on UV exposure from the same patients to evaluate the impact of estimated lifetime sun exposure and sunburns on survival. A trend in the expression of Beclin1 was not observed; however, LC3 expression was increased in melanoma sections compared to normal. Our results provide enticing support that lifetime UVR exposure is associated with autophagy protein expression in melanoma. Citation Format: Kirsten A. M. White, Salina Torres, Todd A. Thompson, Chien-An A. Hu, Orrin Myers, Marianne Berwick. LC3 protein expression associates with UV exposure in melanoma histopathology. [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 2909. doi:10.1158/1538-7445.AM2015-2909

Apoptosis and Autophagy: The Yin–Yang of Homeostasis in Cell Death in Cancer

Apoptosis and autophagy are physiologically necessary pathways that are vital for cell homeostasis. Apoptosis facilitates type I programmed cell death, while the autophagic survival mechanism counteracts apoptosis. Dysregulation in the homeostatic balance between these two essential cellular pathways has been linked to various diseases. We review relevant Janus molecules and their interactomes, as well as lysosomes which play multiple roles in apoptosis and autophagy, and discuss how targeted interventions can be used in cancer prevention and/or therapy.