Melanoma Tumor Development Research Articles

Soy Lunasin and Amaranth Unsaponifiables Loaded in Liposomes Prevented Melanoma Tumor Development by Arresting Cell Cycle, and Upregulating P16, P21, P27, and P53 Inhibitors

Soy Lunasin and Amaranth Unsaponifiables Loaded in Liposomes Prevented Melanoma Tumor Development by Arresting Cell Cycle, and Upregulating P16, P21, P27, and P53 Inhibitors

Abstract 4148: Extracellular matrix biophysical properties change during nevus to melanoma progression

Abstract Cutaneous malignant melanoma is one of the most aggressive forms of skin cancer and its major etiological risk factor is ultraviolet (UV) solar radiation. The extracellular matrix (ECM) has been shown to influence many of the hallmark characteristics of tumor development, survival, and progression across several types of cancer, including melanoma. Interactions between the tumor and the ECM often results in rearrangement, cross-linking, and deposit of specific ECM proteins, which can contribute to stiffening of the tissue and tumorigenesis. To simulate tumorigenesis, we have previously used a hepatocyte growth factor (HGF) transgenic mouse model, which develops melanoma nevii when exposed to UVB radiation. This model is highly representative of human melanoma lesions in terms of biological, genetic, and etiologic criteria. In general, microrheology involves injecting beads into a medium, tracking movement of the beads using two-photon microscopy, and using the movement to calculate the viscosity and elasticity of the medium. The physical properties of the ECM during tumorigenesis have not been well studied at the cellular level, and microrheology has rarely been used in vivo. In our in vivo microrheology study, we injected fluorescent beads into the skin of these transgenic mice. Then, we used intra-vital microscopy with particle tracking to image the beads and measure changes in ECM elasticity as nevii grow into melanoma tumors. These results, combined with transcriptomic analysis of ECM regulation and proteomic analysis of ECM signaling, could provide unique insights into components of the extracellular matrix that are involved in melanoma tumor development and offer opportunities for therapeutics to target oncogenesis. <!–EndFragment–> Citation Format: Aditya Kavuturu, Maira A. Constantino, Antonella Sassano, Glenn Merlino. Extracellular matrix biophysical properties change during nevus to melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4148.

Risk of skin cancers in mycosis fungoides patients receiving PUVA therapy: A real-life experience from a single tertiary center.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma. Skin-directed therapies, including phototherapy, are the first-line treatment modalities. Psoralen plus ultraviolet A light photochemotherapy (PUVA) is quite effective in controlling the disease; however, long-term adverse effects, particularly carcinogenesis, are the cons of this treatment. There are various studies on the negative impact of PUVA on skin cancer in patients with autoimmune skin diseases. The data on the long-term effects of phototherapy on MF patients are scarce. All MF cases that received PUVA alone or combined with other treatments at a single tertiary center were analyzed. This study compared the development of non-melanoma skin cancers, melanoma, and solid organ tumors in MF patients with at least 5-year follow-up data with age- and sex-matched controls. A total of 104 patients were included in the study. Ninety-two malignancies were detected in 16 (15.4%) patients, and six developed multiple malignancies. Skin cancers consisted of 56 basal cell carcinomas, 16 Bowen's disease, four squamous cell carcinomas, three melanomas, two basosquamous cell carcinomas, one Kaposi sarcoma, and one keratoacanthoma were found in nine (8.7%) patients. Eight patients developed three solid cancers and six lymphomas. The risk of developing skin cancer was associated with the total number of PUVA sessions (<250 vs ≥250 sessions; hazard ratio (HR) 4.44, 95% confidence interval (CI) 1.033-19.068; p = .045). 9 (13.2%) of 68 patients who had follow-ups for at least 5 years developed skin cancer. Compared to an age- and sex-matched cohort, the prevalence of new skin cancer was considerably greater (p = .009). Patients with MF are predisposed to develop secondary malignancies, and continual exposure to PUVA may potentiate this risk. Annual digital dermoscopic follow-up in MF patients treated with UVA is advised for early diagnosis and treatment of secondary cutaneous malignancies.

Notch Signaling Suppresses Melanoma Tumor Development in BRAF/Pten Mice.

Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, a prognostic feature in primary melanoma. In this study, we investigated the role of Notch signaling in melanoma tumor development and growth using the genetic model of mouse melanoma by crossing BRAFCA/+/Pten+/+/Tyr-CreER+ (B) and BRAFCA/+/Pten-/-/Tyr-CreER + (BP) mice with Notch1 or Notch2 floxed allele mice. The topical application of tamoxifen induced tumors in BP mice but not in B mice with or without the deletion of either Notch1 or Notch2. These data show that the loss of either Notch1 nor Notch2 can substitute the tumor suppressor function of Pten in BRAFV600E-induced melanomagenesis. However, in Pten-null background, the loss of either Notch1 or Notch2 appeared to accelerate BRAFV600E-induced tumor development, suggesting a tumor suppressor role for Notch1 and Notch2 in BRAFV600E/Pten-null driven melanomagenesis. Quantitative immunochemical analysis of a human cutaneous melanoma tissue microarray that consists of >100 primary tumors with complete clinical history showed a weak to moderate correlation between NOTCH protein levels and clinical and pathological parameters. Our data show that Notch signaling is involved during melanomagenesis and suggest that the identification of genes and signaling pathways downstream of Notch could help devise strategies for melanoma prevention.

Liposomes Loaded with Amaranth Unsaponifiable Matter and Soybean Lunasin Prevented Melanoma Tumor Development Overexpressing Caspase-3 in an In Vivo Model.

The objective of this study was to assess the effectiveness of liposomes loaded with soybean lunasin and amaranth unsaponifiable matter (UM + LunLip) as a source of squalene in the prevention of melanoma skin cancer in an allograft mice model. Tumors were induced by transplanting melanoma B16-F10 cells into the mice. The most effective treatments were those including UM + LunLip, with no difference between the lunasin concentrations (15 or 30 mg/kg body weight); however, these treatments were statistically different from the tumor-bearing untreated control (G3) (p < 0.05). The groups treated with topical application showed significant inhibition (68%, p < 0.05) compared to G3. The groups treated with subcutaneous injections showed significant inhibition (up to 99%, p < 0.05) in G3. During tumor development, UM + LunLip treatments under-expressed Ki-67 (0.2-fold compared to G3), glycogen synthase kinase-3β (0.1-fold compared to G3), and overexpressed caspase-3 (30-fold compared to G3). In addition, larger tumors showed larger necrotic areas (38% with respect to the total tumor) (p < 0.0001). In conclusion, the UM + LunLip treatment was effective when applied either subcutaneously or topically in the melanoma tumor-developing groups, as it slowed down cell proliferation and activated apoptosis.

Liposomes Loaded With Soybean Lunasin and Amaranth Unsaponifiable Matter Promoted Apoptosis Through Caspase 3 and Cell Proliferation Arrest in an In Vivo Melanoma Model

ObjectivesThe objective was to assess the mechanism of action of liposomes loaded with amaranth unsaponifiable matter and lunasin (LpLnUM) in melanoma tumors developed in C57BL/6 mice. MethodsTumors were developed in C57BL/6 using the melanoma cell line B16-F10 and treated every other day for 22 days using LpLnUM with 15 (LpLnUM15) or 30 (LpLnUM30) mg lunasin/kg body weight (BW) via either topically (T) or subcutaneously (S) (treated groups, TG), or lunasin solution at 30 mg/kg BW. Tumors were excised, fixed, and embedded in paraffin, cut with a microtome (5 μm thick), and fixed in microscope slides. For immunohistochemistry, glycogen synthase kinase (GSK)-3β, caspase 3 (C3), and Ki-67 antibodies were used, with horseradish peroxidase and 3,3’-diaminobenzidine as a substrate to reveal protein expression. Hematoxylin and eosin assay assessed tissue structure. Images were captured with a NanoZoomer and analyzed at a 20x magnification. ResultsCompared with the tumor-untreated control (UC), T and S applied liposomes inhibited the tumor in 61 and 71%, respectively. No difference between lunasin concentrations (p < 0.05). The smaller the tumor volume, the higher C3 expression (r = –0.819, p < 0.05). Tumors showed a C3 overexpression in those groups S treated with LpLnUM15 (1.41-fold, p < 0.01) and LpLnUM30 (1.53-fold, p < 0.01) compared with the UC. In contrast, lunasin solution (without liposomes) T applied at 30 mg/kg BW had no difference with the UC, but it was different (p < 0.05) when compared with the groups treated with only S applied liposomes. GSK-3β suggested that even though the expression decreased in the TG there was no difference among controls and treatments. The proliferation marker Ki-67 showed an under expression of the marker between the TG (p < 0.001) and the UC. The Ki-67 expression in mice T treated with LpLnUM30 and LpLnUM15 was inhibited 89 and 76%, respectively. In those mice with S application, the Ki-67 inhibition was 81 and 93% when treated with LpLnUM30 and LpLnUM15, respectively. These last results suggest an arrest in the cell cycle. ConclusionsLpLnUM15 and LpLnUM30 prevented melanoma tumor development by promoting cell apoptosis via C3 expression and cell cycle arrest regardless of lunasin concentration and application method in mice. Funding SourcesUSDA National Institute of Food and Agriculture, CONACyT fellowship.

FBXO32 links ubiquitination to epigenetic reprograming of melanoma cells

Ubiquitination by serving as a major degradation signal of proteins, but also by controlling protein functioning and localization, plays critical roles in most key cellular processes. Here, we show that MITF, the master transcription factor in melanocytes, controls ubiquitination in melanoma cells. We identified FBXO32, a component of the SCF E3 ligase complex as a new MITF target gene. FBXO32 favors melanoma cell migration, proliferation, and tumor development in vivo. Transcriptomic analysis shows that FBXO32 knockdown induces a global change in melanoma gene expression profile. These include the inhibition of CDK6 in agreement with an inhibition of cell proliferation and invasion upon FBXO32 silencing. Furthermore, proteomic analysis identifies SMARC4, a component of the chromatin remodeling complexes BAF/PBAF, as a FBXO32 partner. FBXO32 and SMARCA4 co-localize at loci regulated by FBXO32, such as CDK6 suggesting that FBXO32 controls transcription through the regulation of chromatin remodeling complex activity. FBXO32 and SMARCA4 are the components of a molecular cascade, linking MITF to epigenetics, in melanoma cells.

MYH9 suppresses melanoma tumorigenesis, metastasis and regulates tumor microenvironment.

Non-muscle myosin IIA heavy chain (MYH9) has been implicated in many physiological and pathological functions including cell adhesion, polarity, motility to cancer. However, its role in melanoma remains unexplored. The aim of our study was to evaluate the role of MYH9 in melanoma tumor development and metastasis and further to find out the potential underlying mechanisms. In this study, we evaluated the in vitro migratory and invasive properties and in vivo tumor development and metastasis in C57BL/6 mice by silencing MYH9 in B16F10 melanoma cells. Knocking down MYH9 enhanced migration and invasiveness of B16F10 cells in vitro. Furthermore, MYH9 silencing accelerated tumor growth and metastasis in melanoma subcutaneous and intravenous mouse models. Next, oncogenes analysis revealed epithelial-mesenchymal transition and Erk signaling pathway are being regulated with MYH9 expression. Finally, MYH9 silencing in B16F10 cells modulates the tumor microenvironment by manipulating the leukocytes and macrophages infiltration in tumors. These findings established the opposing role of MYH9 as a tumor suppressor in melanoma suggesting specific MYH9 based approaches in therapeutics.

Salubrinal in Combination With 4E1RCat Synergistically Impairs Melanoma Development by Disrupting the Protein Synthetic Machinery.

Increased protein synthesis is a key process in melanoma, which is regulated by the ALDH18A1 gene encoding pyrroline-5-carboxylate synthase (P5CS). P5CS is involved in proline biosynthesis and targeting ALDH18A1 has previously been shown to inhibit melanoma development by decreasing intracellular proline levels to increase the phosphorylation of eIF2α mediated by GCN2, which then impairs mRNA translation. Since there are no current inhibitors of P5CS, decreased eIF2α phosphorylation in melanoma was targeted using salubrinal (a specific inhibitor of eIF2α phosphatase enzymes). While salubrinal alone was ineffective, the combined use of salubrinal and 4E1RCat (a dual inhibitor of eIF4E:4E-BP1 and eIF4E:eIF4G interaction to prevent assembly of the eIF4F complex and inhibit cap-dependent translation) was found to be effective at decreasing protein synthesis, protein translation, and cell cycle progression to synergistically decrease melanoma cell viability and inhibited xenograft melanoma tumor development. The combination of these agents synergistically decreased melanoma cell viability while having minimal effect on normal cells. This is the first report demonstrating that it is possible to inhibit melanoma viability by targeting eIF2α signaling using salubrinal and 4E1RCat to disrupt assembly of the eIF4F complex.

ADORA1 Inhibition Promotes Tumor Immune Evasion by Regulating the ATF3-PD-L1 Axis

Here, we show that tumor ADORA1 deletion suppresses cell growth in human melanoma cell lines invitro and tumor development invivo in immune-deficient xenografts. However, this deletion induces the upregulation of PD-L1 levels, which inactivates cocultured Tcells invitro, compromises anti-tumor immunity invivo, and reduces anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of ADORA1-deficient or ADORA1 antagonist-treated melanoma and NSCLC immune-competent mouse models. Mechanistically, we identify ATF3 as the factor transcriptionally upregulating PD-L1 expression. Tumor ATF3 deletion improves the effect of ADORA1 antagonist treatment of melanoma and NSCLC xenografts. We observe higher ADORA1, lower ATF3, and lower PD-L1 expression levels in tumor tissues from nonresponders among PD-1 mAb-treated NSCLC patients.

Thymosin β4 Regulates Focal Adhesion Formation in Human Melanoma Cells and Affects Their Migration and Invasion.

Thymosin β4 (Tβ4), a multifunctional 44-amino acid polypeptide and a member of actin-binding proteins (ABPs), plays an important role in developmental processes and wound healing. In recent years an increasing number of data has been published suggesting Tβ4’s involvement in tumorigenesis. However, Tβ4’s role in melanoma tumor development still remains to be elucidated. In our study we demonstrate that Tβ4 is crucial for melanoma adhesion and invasion. For the purpose of our research we tested melanoma cell lines differing in invasive potential. Moreover, we applied shRNAs to silence TMSB4X (gene encoding Tβ4) expression in a cell line with high TMSB4X expression. We found out that Tβ4 is not only a component of focal adhesions (FAs) and interacts with several FAs components but also regulates FAs formation. We demonstrate that Tβ4 level has an impact on FAs’ number and morphology. Moreover, manipulation with TMSB4X expression resulted in changes in cells’ motility on non-coated and MatrigelTM (resembling basement membrane composition)-coated surfaces and drastically decreased invasion abilities of the cells. Additionally, a correlation between Tβ4 expression level and exhibition of mesenchymal-like [epithelial-mesenchymal transition (EMT)] features was discovered. Cells with lowered TMSB4X expression were less EMT-progressed than control cells. Summarizing, obtained results show that Tβ4 by regulating melanoma cells’ adhesion has an impact on motility features and EMT. Our study not only contributes to a better understanding of the processes underlying melanoma cells’ capacity to create metastases but also highlights Tβ4 as a potential target for melanoma management therapy.

Factors Affecting Growth Kinetics and Spontaneous Metastasis in the B16F10 Syngeneic Murine Melanoma Model.

Melanoma is an immunogenic tumor that can metastasize quickly to proximal and distal sites, thus complicating the application of therapeutic modalities. Numerous mouse model systems have been used to gain understanding of the immunobiology and metastatic potential of melanoma. Here, we report the optimization of a syngeneic mouse melanoma model protocol using the mouse B16-derived melanoma cell line B16F10 that ensures the production of tumors on mice pinnae that are similar in size between animals and that enlarge in a time-dependent manner. In this model, B16F10 cells are first allowed to develop tumors after injection in the interscapular area or flank of C57BL/6J mice. Subsequently, the tumors are harvested, cells dissociated and injected into mouse pinnae. Dose-dependent studies revealed that injection of 2 × 105 cells allowed for slow tumor enlargement, producing tumors averaging 100 mm³ within 2 to 3 wk with a metastatic frequency of 100%. This experimental protocol will be useful in dissecting the immunobiology of melanoma tumor development and metastasis and the evaluation of immunotherapeutic antimelanoma therapies.

Deficiency of parkin suppresses melanoma tumor development and metastasis through inhibition of MFN2 ubiquitination

Parkin, a critical gene of Parkinson's disease, is involved in the development of numerous cancers. However, the effect of parkin deficiency on melanoma growth and metastasis has not been reported. We showed that the tumor size and number of surface lung metastases, and expression of tumor growth and metastasis marker proteins were significantly lower in parkin-KO mice than those observed in non-transgenic controls. In an in vitro study, we also showed that parkin siRNA inhibited cell growth and migration of B16F10 and SK-Mel-28 cells. Parkin-specific ubiquitination of mitofusin-2 (MFN2) was decreased in tumors and metastasized lung tissues of parkin-KO mice. Moreover, we showed that parkin directly binds and ubiquitinates MFN2. Knockdown of MFN2 decreased the expression of Bax and apoptotic cell death, but increased that of Bcl2 and apoptotic cancer cell death. However, these effects were reversed by knockdown of parkin. Conversely, inhibitory effects on melanoma growth and migration of parkin siRNA were reversed by MFN2 siRNA. These data indicate that melanoma development was inhibited in parkin-KO mice through maintaining of MFN2 level by inhibition of ubiquitinating ability of parkin.

EPAC-RAP1 Axis-Mediated Switch in the Response of Primary and Metastatic Melanoma to Cyclic AMP.

Cyclic AMP (cAMP) is an important second messenger that regulates a wide range of physiologic processes. In mammalian cutaneous melanocytes, cAMP-mediated signaling pathways activated by G-protein-coupled receptors (GPCR), like melanocortin 1 receptor (MC1R), play critical roles in melanocyte homeostasis including cell survival, proliferation, and pigment synthesis. Impaired cAMP signaling is associated with increased risk of cutaneous melanoma. Although mutations in MAPK pathway components are the most frequent oncogenic drivers of melanoma, the role of cAMP in melanoma is not well understood. Here, using the Braf(V600E)/Pten-null mouse model of melanoma, topical application of an adenylate cyclase agonist, forskolin (a cAMP inducer), accelerated melanoma tumor development in vivo and stimulated the proliferation of mouse and human primary melanoma cells, but not human metastatic melanoma cells in vitro The differential response of primary and metastatic melanoma cells was also evident upon pharmacologic inhibition of the cAMP effector protein kinase A. Pharmacologic inhibition and siRNA-mediated knockdown of other cAMP signaling pathway components showed that EPAC-RAP1 axis, an alternative cAMP signaling pathway, mediates the switch in response of primary and metastatic melanoma cells to cAMP. Evaluation of pERK levels revealed that this phenotypic switch was not correlated with changes in MAPK pathway activity. Although cAMP elevation did not alter the sensitivity of metastatic melanoma cells to BRAF(V600E) and MEK inhibitors, the EPAC-RAP1 axis appears to contribute to resistance to MAPK pathway inhibition. These data reveal a MAPK pathway-independent switch in response to cAMP signaling during melanoma progression.Implications: The prosurvival mechanism involving the cAMP-EPAC-RAP1 signaling pathway suggest the potential for new targeted therapies in melanoma. Mol Cancer Res; 15(12); 1792-802. ©2017 AACR.

Expression signatures of early-stage and advanced medaka melanomas

Melanoma is one of the most aggressive tumors with a very low survival rate once metastasized. The incidence of newly detected cases increases every year suggesting the necessity of development and application of innovative treatment strategies. Human melanoma develops from melanocytes localized in the epidermis of the skin to malignant tumors because of deregulated effectors influencing several molecular pathways. Despite many advances in describing the molecular changes accompanying melanoma formation, many critical and clinically relevant molecular features of the transformed pigment cells and the underlying mechanisms are largely unknown. To contribute to a better understanding of the molecular processes of melanoma formation, we use a transgenic medaka melanoma model that is well suited for the investigation of melanoma tumor development because fish and human melanocytes are both localized in the epidermis. The purpose of our study was to gain insights into melanoma development from the first steps of tumor formation up to melanoma progression and to identify gene expression patterns that will be useful for monitoring treatment effects in drug screening approaches. Comparing transcriptomes from juvenile fish at the tumor initiating stage with nevi and advanced melanoma of adults, we identified stage specific expression signatures and pathways that are characteristic for the development of medaka melanoma, and are also found in human malignancies.

Identifying the structure-activity relationship of leelamine necessary for inhibiting intracellular cholesterol transport.

Leelamine is an anticancer chemotherapeutic agent inhibiting intracellular cholesterol transport. Cell death mediated by leelamine occurs due to the lysosomotropic property of the compound, its accumulation in the lysosome, and inhibition of cholesterol transport leading to lack of availability for key processes required for functioning of cancer cells. The present study dissects the structure-activity-relationship of leelamine using synthesized derivatives of leelamine and abietic acid, a structurally similar compound, to identify the moiety responsible for anti-cancer activity. Similar to leelamine, all active derivatives had an amino group or a similar moiety that confers a lysosomotropic property to the compound enabling its accumulation in the lysosome. Active derivatives inhibited intracellular cholesterol transport and hindered xenografted melanoma tumor development without obvious systemic toxicity. In silico studies suggested that active derivatives accumulating in lysosomes bound to NPC1, a protein responsible for cholesterol export from the lysosome, to inhibit its activity that then caused accumulation, and lack of cholesterol availability for other key cellular activities. Thus, active derivatives of leelamine or abietic acid maintained lysosomotropic properties, bound to NPC1, and disrupted cellular cholesterol transport as well as availability to retard tumor development.

Synergistic inhibitory effects of Celecoxib and Plumbagin on melanoma tumor growth

Melanoma is a highly drug resistant cancer. To circumvent this problem, a class of synergistically acting drug combinations, which inhibit multiple key pathways in melanoma cells, could be used as one approach for long-term treatment of this deadly disease. A screen has been undertaken on cell lines to identify those that could be combined to synergistically kill melanoma cells. Plumbagin and Celecoxib are two agents that were identified to synergistically kill melanoma cells by inhibiting the COX-2 and STAT3 pathways, which are constitutively activated in up to 70% of melanomas. The combination of these two drugs was more effective at killing melanoma cells than normal cells and decreased cellular proliferation as well as induced apoptosis of cultured cells. The drug combination inhibited development of xenograft melanoma tumors by up to 63% without affecting animal weight or blood biomarkers of organ function, suggesting negligible toxicity. Mechanistically, combination of Celecoxib and Plumbagin decreased melanoma cell proliferation and retarded vascular development of tumors mediated by inhibition of COX-2 and STAT3 leading to decreased levels of key cyclins key on which melanoma cell were dependent for survival.

Abstract 4623: Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance

Abstract Notch is one of the simplest signaling pathways that is required for almost every cell type in the body. It plays several roles such as cell fate decision, differentiation and stem cell maintenance. Notch signaling plays a role in the maintenance of melanoblasts and melanocyte stem cells of the epidermis by preventing their apoptosis. Notch signaling has also been implicated in different aspects of melanoma biology including tumor initiation, progression and metastasis. Accordingly, Notch has been targeted for melanoma therapy both in preclinical xenograft models as well as clinical trials. Notch inhibitors, mainly γ-secretase inhibitors, have been widely used for this purpose. However, clinical trials with these agents have not been proven to be highly effective for melanoma treatment. In previously published studies we showed that Notch signaling is involved in regulation of melanoma trans-differentiation, a prognostic feature in primary melanoma. In this study, we reevaluated the role of Notch in melanoma development, cell survival and sensitivity to BRAF and MEK inhibitors. First, our preliminary data using the BRAFV600E/Pten mouse model showed that topical application of γ-secretase inhibitor DAPT delays tumor formation and growth. In several early passage metastatic human melanoma cell lines, we found that Notch receptors (Notch 1-3) are highly upregulated compared to normal epidermal melanocytes. Interestingly, mRNA levels for Notch1-3 receptors in normal melanocytes were either higher or comparable to their levels in the cell lines. This suggested that Notch receptor levels are tightly regulated in normal melanocytes by post-transcriptional mechanisms. Presence of cleaved Notch intracellular domain (NICD), both cytoplasmic and nuclear, in melanoma cells, but not in melanocytes, indicated constitutive activation of Notch signaling in melanoma cells. However, inhibition of Notch activation by treatment with γ-secretase inhibitor had no significant effect on survival of melanoma cells in vitro. Thus, Notch signaling appears to be dispensable for melanoma cell survival. Additionally, inhibition of Notch activation did not alter the sensitivity of melanoma cells to BRAF(V600E) and MEK inhibitors. Our results highlight the need for re-examination of Notch-regulated melanoma trans-differentiation as a therapeutic outcome. Citation Format: Dareen Mikheil, Carlos Rodriguez, Ashika Jayanthi, Prithvi Yarlagadda, Vijaysaradhi Setaluri. Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance. [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 4623.

618 Topical application of cyclic AMP modulators regulates melanoma tumor development and tumor growth

618 Topical application of cyclic AMP modulators regulates melanoma tumor development and tumor growth

Highlights of This Issue

Tenkerian and colleagues address the cross-regulation between the mTOR and eIF2 kinase pathways and the importance of these mechanisms for cell survival. The key finding is that mTORC2 can repress PERK through the activation of Akt/PKB and phosphorylation of an inhibitory PERK site (Thr-799), which interferes with the activity of this eIF2 kinase. This mechanism provides new insight into the cross-talk between the mTOR and eIF2 kinase pathways and provides an explanation for why loss of TSC function can induce eIF2 phosphorylation. These ideas are important for the development of new treatment strategies for many cancers.The development of agents that selectively potentiate radiation and chemotherapy in DNA repair-deficient cancers is an attractive direction for cancer therapy. Stachelek and colleagues describe a novel series of such compounds identified by a high-throughput synthetic lethality screen. The compounds contain an aryl sulfonamide motif and strongly sensitize BRCA2-deficient tumors to DNA-damaging therapy. By themselves, they also induce synthetic lethality in cancer cells deficient in DNA repair-related genes, including BRCA2 and PTEN. This new class of agents also offers the possibility of heightened treatment efficacy with reduced toxicity through synergism with other targeted agents, such as PARP inhibitors.The importance of protein translation and the possibility of therapeutically targeting this process in cancer cells are often overlooked. In the study by Kardos and colleagues, the significance of targeting protein translation in cancer by disrupting the synthesis of the amino acid proline is demonstrated. The study shows that melanoma cells require high proline concentrations for survival and are sensitive to disruption of these levels. Targeting aldehyde dehydrogenase (ALDH18A1), a key point in proline biosynthesis pathways, can be used to retard melanoma tumor development by activating the ser/thr protein kinase GCN2 (general control nonderepressible 2), which phosphorylates eIF2 causing downregulation of protein synthesis.Reduced expression of the translation initiation factor eIF3e has been observed in both breast and lung tumors. Previous study has suggested that reduced eIF3e expression in breast cancer cells causes epithelial-to-mesenchymal transition (EMT), an event that drives tumor metastasis. Desnoyers and colleagues demonstrate that reduced eIF3e expression also causes EMT in non-small cell lung cancer (NSCLC). This reduced eIF3e expression leads to overproduction of the TGFβ cytokine, which is known to potently induce EMT. Moreover, inhibition of TGFβ signaling in NSCLC cells with reduced eIF3e expression reverses EMT, highlighting the potential to use TGFβ inhibition as a therapeutic for NSCLC patients whose tumors display reduced eIF3e expression.