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We present an integrated study to understand the key role of senescent fibroblasts in driving melanoma progression. Based on the hybrid cellular automata paradigm, we developed an in silico model of normal skin. The model focuses on key cellular and microenvironmental variables that regulate interactions among keratinocytes, melanocytes, and fibroblasts, key components of the skin. The model recapitulates normal skin structure and is robust enough to withstand physical as well as biochemical perturbations. Furthermore, the model predicted the important role of the skin microenvironment in melanoma initiation and progression. Our in vitro experiments showed that dermal fibroblasts, which are an important source of growth factors in the skin, adopt a secretory phenotype that facilitates cancer cell growth and invasion when they become senescent. Our coculture experiments showed that the senescent fibroblasts promoted the growth of nontumorigenic melanoma cells and enhanced the invasion of advanced melanoma cells. Motivated by these experimental results, we incorporated senescent fibroblasts into our model and showed that senescent fibroblasts transform the skin microenvironment and subsequently change the skin architecture by enhancing the growth and invasion of normal melanocytes. The interaction between senescent fibroblasts and the early-stage melanoma cells leads to melanoma initiation and progression. Of microenvironmental factors that senescent fibroblasts produce, proteases are shown to be one of the key contributing factors that promoted melanoma development from our simulations. Although not a direct validation, we also observed increased proteolytic activity in stromal fields adjacent to melanoma lesions in human histology. This leads us to the conclusion that senescent fibroblasts may create a prooncogenic skin microenvironment that cooperates with mutant melanocytes to drive melanoma initiation and progression and should therefore be considered as a potential future therapeutic target. Interestingly, our simulations to test the effects of a stroma-targeting therapy that negates the influence of proteolytic activity showed that the treatment could be effective in delaying melanoma initiation and progression. Cancer Res; 73(23); 6874–85. ©2013 AACR.

Uveal melanoma is the most common adult cancer of the eye. Although uveal melanoma has a low incidence, it has a high capability to develop metastases, mainly to the liver. Mortality rates are high and new therapeutic strategies are urgently needed. 85-90% of all uveal melanomas harbor driver mutations in GNAQ or GNA11 leading to constitutive activation of many signaling pathways, including the MAPK pathway. Although MEK inhibitors have been evaluated clinically for metastatic uveal melanoma, responses are short-lived, and the mechanisms of adaptation are not well delineated. In the current study we performed mass spectrometry-based proteomics to define the adaptive response of uveal melanoma cells to MEK inhibition at a systems level with the hope of identifying the key escape pathways. These analyses identified MEK inhibition to lead to increased histone phosphorylation and histone deacetylase (HDAC) activity, as well as reorganization of the cytoskeleton associated with cortactin phosphorylation. MEK inhibition also increased receptor tyrosine kinase (RTK) signaling (through ROR1 and IGFIR) and enhanced AKT phosphorylation. Use of pan-HDAC inhibitors sensitized the uveal melanoma cells to MEK inhibition leading to enhanced apoptosis and decreased cell invasion. Mechanistic studies showed the MEK+HDAC inhibitor combination inhibited MAPK, PI3K/AKT and the Hippo signaling, an effect associated with increased cell death inhibition of cell invasion. Chronic MEK inhibitor treatment led to acquired resistance that was associated with increased RTK signaling and the adoption of an invasive phenotype. Studies are ongoing to identify the key HDAC isoform required for the adaptation to MEK inhibitor therapy as well as the anti-invasive potential of the cortactin knockdown with the goal of developing novel combination therapies that can be evaluated clinically. Citation Format: Fernanda Faião-Flores, Silvya S. Maria-Engler, Zeynep Eroglu, Keiran S. Smalley. Adaptation of uveal melanoma cells to MEK inhibition can be overcome through HDAC inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4814.

Cervical cancer is related to the persistent infection by high-risk human papillomavirus (HPV) subtypes, such as types 16 and 18. The E6 and E7 oncoproteins significantly contribute to the carcinogenic potential of high-risk HPV, through the degradation of p53 and pRb, respectively. The exoprotease dipeptidyl peptidase IV (DPPIV) also known as CD26, is a multifunctional protein involved in several processes related to cancer. DPPIV/CD26 has an intrinsic peptidase activity, able to degrade some bioactive peptides and chemokines, thereby regulating cell migration, proliferation and survival. It has been reported that the reduction in DPPIV/CD26 expression is associated with tumor progression in different types of cancer. Moreover, studies in cervical cancer samples and cell lines point to a reduction in the expression of this protein. However, there are no studies addressing the relationship between HPV infection and DPPIV/CD26 expression. We analyzed the influence of HPV oncoproteins on DPPIV/CD26 expression in human keratinocyte transduced with E6, E7 and E6E7. We observed that HPV16 E6 expression is associated to a reduction in DPPIV/CD26 expression at mRNA and protein levels, whereas HPV16 E7 does not affect DPPIV/CD26 expression. HPV16 E6E7 co-expression leads to a reduction in DPPIV/CD26 expression similar to that found in E6 transduced cells. In addition, we note a more pronounced reduction in DPPIV/CD26 expression in immortalized cells expressing E6E7. Furthermore, keratinocytes expressing HPV16 E6 mutant sequence and HPV11 E6, that do not bind to E6AP and do not degrade p53, have no effect on DPPIV/CD26 expression. Our results suggest that the HPV16 E6 oncoprotein can downregulates DPPIV/CD26. In addition, preliminary results suggest that modulation of DPPIV/CD6 mediated by E6 can be dependent on degradation of p53, since HPV16 E6 mutant and low-risk HPV E6 were not able to affect CD26 expression. Furthermore, the process of cell immortalization also appears to be related to the reduction of DPPIV/CD26 expression. Therefore, these results indicate that HPV oncoproteins promote imbalance DPPIV/CD26 expression which may be relate to cervical carcinogenesis. Citation Format: Aline Beckenkamp, Bruna Prati, Silvya Stuchi Maria-Engler, Guido Lenz, Diogo André Pilger, Enrique Mario Pierulivo, Andréia Buffon. HPV16 E6 oncoprotein modulates the DPPIV/CD26 in primary keratinocytes [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 5341. doi:10.1158/1538-7445.AM2017-5341

Melanoma represents the deadliest of all skin cancers being responsible for more than 75% of skin cancer-related deaths. Although the results with BRAF inhibitors have been very promising, practically all of the patients treated thus far have developed resistance to these drugs. The Hedgehog (Hh) signaling pathway has been implicated in a variety of malignancies, including melanoma. However, there are no reports on the activation of this pathway in the setting of vemurafenib-resistance. Herein, we first identified that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, showed increased levels of Hh pathway component genes glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared to naïve cells. We also observed these findings in melanoma samples from patients. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with non-canonical Hh signaling, involving TGFβ/SMAD signaling. Knockdown of GLI1 and GLI2 restored the vemurafenib-resistant cells to sensitivity, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a 3D skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphtalmia transcription factor (MITF) upregulation. Our findings also demonstrated that GLI1/GLI2 modulation could be a useful strategy to prevent drug resistance. Alternating pre-treatment with vemurafenib and Gant61 significantly reduced IC50 of subsequent vemurafenib treatment in naïve melanoma cells, demonstrating a promising approach for the control of vemurafenib-resistance in patients with unresectable melanoma. The modulation of vemurafenib chemosensitivity was triggered by GLI1/GLI2 repression during the acquisition of resistance, which did not occur in the treatment protocols with only vemurafenib continuously or in alternate days. The alternating treatment regimen with vemurafenib and Gant61 was able to suppress GLI expression, delaying or decreasing vemurafenib resistance. Taken together, our data demonstrated an unprecedented mechanism of vemurafenib resistance by GLI1/GLI2 upregulation, shedding light on the development of Hh pathway inhibitors as a promising strategy for melanoma treatment. Citation Format: Fernanda Faiao-Flores, Debora Kristina Magalhaes Alves-Fernandes, Paula Comune Pennacchi, Silvana Sandri, Anna Luiza Silva Almeida Vicente, Cristovam Scapulatempo-Neto, Vinicius de Lima Vazquez, Rui Manuel Reis, Keiran S. Smalley, Silvya Stuchi Maria-Engler. Targeting hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells. [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 4669.

Strategies for patients whose melanomas are BRAF wild-type are completely lacking. We here report on the mechanisms underlying the long-term responses (>12 months) of 2 BRAF wild-type (WT) melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. A panel of 7 BRAF/NRAS-WT cell lines was leveraged to investigate strategies to overcome chemoresistance. Electron microscopy, western blotting and flow cytometry assays were integrated to simultaneously follow fluctuations in autophagic flux, reactive oxygen species levels and cytotoxicity caused from chronic treatment of cells with chemotherapy, MK-2206, lysomotropic agents and/or anti-oxidants. MK-2206 potently inhibited phospho-AKT signaling, but did not impact melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin induced little apoptosis following 3-day treatment, but led to cytotoxicity in long-term colony formation and 3D spheroid assays. Mechanistically the combination was noted to induce autophagy and disrupt reactive oxygen species homeostasis, as shown by increased LC3-II expression, the formation of LC3 punctae, acridine orange protonation, the accumulation of autophagosomes and increased ROS detection using DHR-123. At earlier time points, autophagy was protective with cytotoxicity enhanced following chloroquine and bafilomycin treatment as well as the increased cytotoxic effects seen in ATG5-/- MEFs. Although prolonged MK-2206/chemotherapy treatment (6-9 days) led to high levels of autophagy induction and a switch to caspase-dependent apoptosis, drug resistant clones still eventually emerged. The importance of autophagy for the escape process was suggested by the lack of ATG5-/- MEF colonies seen following combination therapy treatment. Autophagy inhibition enhanced the cell death response through increased generation of ROS and could be reversed by anti-oxidants. In summary, we demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma. We further show that the administration of an autophagy inhibitor may prevent resistance from emerging to this drug combination. Citation Format: Vito W. Rebecca, Renato Massaro, Inna Fedorenko, Geoffrey T. Gibney, Vernon K. Sondak, Ravi K. Amaravadi, Shengkan Jin, Silvya S. Maria-Engler, Ragini Kudchadkar, Keiran S. M. Smalley. BRAF wild-type melanoma survival hinges upon AKT activity and adaptive autophagic reprogramming in response to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 809. doi:10.1158/1538-7445.AM2014-809

Although targeted therapy strategies are currently lacking for patients whose melanomas are BRAF wild-type, some therapeutic efficacy was noted in a phase I clinical trial of the AKT inhibitor MK-2206 in combination with Carboplatin (C) and Paclitaxel (P). In the present study we addressed the mechanism underlying the responses to MK-2206 + CP in BRAF wild-type melanoma. As single agents, MK-2206, carboplatin and paclitaxel had only limited cytotoxic activity in both 2D and 3D cell culture and induced very little apoptosis. Prolonged treatment (>9 days) with the AKT inhibitor in combination with C+P led to significant levels of cell death in 4 3D spheroid culture models of BRAF-wild type melanoma. In contrast, treatment with single agent MK-2206 or C+P alone induced little cell death. To investigate the mechanism of interaction between these three compounds we focused upon autophagy induction. It was noted that significant levels of autophagy, as demonstrated by increased LC3-II staining, p62 expression and increased acridine orange staining, were induced following up to 6 days of treatment with the combination. In this context autophagy was protective, with levels of cell death induced by the combination significantly enhanced following treatment with the autophagy inhibitor chloroquine. The protective autophagy induced by MK-2206 + CP was only transient, with diminished levels of autophagy being observed by 9 days. The observed reduction in autophagic flux was associated with marked increases in cell death. In summary it appears that chronic insult with MK-2206 and CP pushes autophagy to its limit, relinquishing its protective role and enhancing sensitivity of the cells to therapy-induced apoptosis. These studies form the basis for a phase II trial evaluating MK-2206 in combination with chemotherapy, as well as autophagy inhibitors, in patients whose melanomas are BRAF-wild type. Citation Format: Vito W. Rebecca, Renato R. Massaro, Silvya S. Maria-Engler, Jane L. Messina, Geoffrey T. Gibney, Vernon K. Sondak, Ragini Kudchadkar, Keiran S. Smalley. Exhaustion of autophagy-mediated cell survival underlies the pre-clinical and clinical activity of chemotherapy combined with the AKT inhibitor MK-2206 in BRAF wild-type melanoma. [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 1659. doi:10.1158/1538-7445.AM2013-1659