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Bidirectional signalling between the tumour and stroma shapes tumour aggressiveness and metastasis. ATF4 is a major effector of the Integrated Stress Response, a homeostatic mechanism that couples cell growth and survival to bioenergetic demands. Using conditional knockout ATF4 mice, we show that global, or fibroblast-specific loss of host ATF4, results in deficient vascularization and a pronounced growth delay of syngeneic melanoma and pancreatic tumours. Single-cell transcriptomics of tumours grown in Atf4 Δ/Δ mice uncovered a reduction in activation markers in perivascular cancer-associated fibroblasts (CAFs). Atf4 Δ/Δ fibroblasts displayed significant defects in collagen biosynthesis and deposition and a reduced ability to support angiogenesis. Mechanistically, ATF4 regulates the expression of the Col1a1 gene and levels of glycine and proline, the major amino acids of collagen. Analyses of human melanoma and pancreatic tumours revealed a strong correlation between ATF4 and collagen levels. Our findings establish stromal ATF4 as a key driver of CAF functionality, malignant progression and metastasis., (© 2022. The Author(s).)

Tumor-derived extracellular vesicles (TEV) "educate" healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The anti-tumorigenic effects that type 1 interferons (IFN1) elicited in the in vitro studies prompted consideration of IFN1 as a potent candidate for clinical treatment. Though not all patients responded to IFN1, clinical trials have shown that patients with high risk melanoma, a highly refractory solid malignancy, benefit greatly from intermediate IFN1 treatment in regards to relapse-free and distant-metastasis-free survival. The mechanisms by which IFN1 treatment at early stages of disease suppress tumor recurrence or metastatic incidence are not fully understood. Intracellular IFN1 signaling is known to affect cell differentiation, proliferation, and apoptosis. Moreover, recent studies have revealed specific IFN1-regulated genes that may contribute to IFN1-mediated suppression of cancer progression and metastasis. In concert, expression of these different IFN1 stimulated genes may impede numerous mechanisms that mediate metastatic process. Though, IFN1 treatment is still utilized as part of standard care for metastatic melanoma (alone or in combination with other therapies), cancers find the ways to develop insensitivity to IFN1 treatment allowing for unconstrained disease progression. To determine how and when IFN1 treatment would be most efficacious during disease progression, we must understand how IFN1 signaling affects different metastasis steps. Here, we specifically focus on the anti-metastatic role of endogenous IFN1 and parameters that may help to use pharmaceutical IFN1 in the adjuvant treatment to prevent cancer recurrence and metastatic disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The unfolded protein response (UPR) regulates cell fate following exposure of cells to endoplasmic reticulum stresses. PERK, a UPR protein kinase, regulates protein synthesis and while linked with cell survival, exhibits activities associated with both tumor progression and tumor suppression. For example, while cells lacking PERK are sensitive to UPR-dependent cell death, acute activation of PERK triggers both apoptosis and cell cycle arrest, which would be expected to contribute tumor suppressive activity. We have evaluated these activities in the BRAF-dependent melanoma and provide evidence revealing a complex role for PERK in melanoma where a 50% reduction is permissive for BrafV600E-dependent transformation, while complete inhibition is tumor suppressive. Consistently, PERK mutants identified in human melanoma are hypomorphic with dominant inhibitory function. Strikingly, we demonstrate that small molecule PERK inhibitors exhibit single agent efficacy against BrafV600E-dependent tumors highlighting the clinical value of targeting PERK., Competing Interests: KAS and MCGP are employed by Eli Lilly.

Oncogene activation induces DNA damage responses and cell senescence. We report a key role of type I interferons (IFNs) in oncogene-induced senescence. IFN signaling-deficient melanocytes expressing activated Braf do not exhibit senescence and develop aggressive melanomas. Restoration of IFN signaling in IFN-deficient melanoma cells induces senescence and suppresses melanoma progression. Additional data from human melanoma patients and mouse transplanted tumor models suggest the importance of non-cell-autonomous IFN signaling. Inactivation of the IFN pathway is mediated by the IFN receptor IFNAR1 downregulation that invariably occurs during melanoma development. Mice harboring an IFNAR1 mutant, which is partially resistant to downregulation, delay melanoma development, suppress metastatic disease, and better respond to BRAF or PD-1 inhibitors. These results suggest that IFN signaling is an important tumor-suppressive pathway that inhibits melanoma development and progression and argue for targeting IFNAR1 downregulation to prevent metastatic disease and improve the efficacy of molecularly target and immune-targeted melanoma therapies., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Interferon alpha (IFNalpha) is widely used in treatment of malignant melanoma patients. This cytokine acts on cells by engaging Type I IFN receptor consisting of two subunits, (IFNAR1 and IFNAR2) followed by activation of Janus kinases (Jak). Levels of IFNAR1 (regulated via degradation mediated by the betaTrcp E3 ubiquitin ligase) and IFNalpha signaling were reduced in 1205Lu melanoma cell line that harbors activated BRAF and exhibits high levels of betaTrcp ubiquitin ligase. Expression of stabilized IFNAR1 in melanoma cells decreased their tumorigenicity. Furthermore, RNAi-mediated BRAF knockdown and pharmacologic inhibition of either Raf or MEK1 decreased levels of betaTrcp and stabilized IFNAR1. However, despite causing stabilization of IFNAR1, Raf inhibitor BAY 43-9006 interfered with cellular responses to IFNalpha most likely due to its ability to directly inhibit Jak activity. We discuss the implications of this result for combination therapy with BAY 43-9006 and IFNalpha in melanoma patients.

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor kappaB (NF-kappaB) transcription factor activation triggered by proteolysis of its inhibitor IkappaB. IkappaB degradation is dependent upon its phosphorylation by the IkappaB kinase (IKK) complex and subsequent ubiquitination facilitated by beta-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF(V600E) exhibit enhanced beta-Trcp expression, increased IKK activity and a concomitant increase in the rate of IkappaBalpha degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAF(V600E) expression by RNA interference in human melanoma cells leads to decreased IKK activity and beta-Trcp expression, stabilization of IkappaB, inhibition of NF-kappaB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-kappaB activity and to increased survival of melanoma cells.

Malignant melanoma is similar to the other types of cancer in terms that the pathogenesis of this lethal disease includes abnormal activation of proteins that mediate oncogenic signaling as well as inhibition of anti-proliferative and pro-apoptotic protein regulators. Activity of both types of cellular regulators is often dependent on their abundance and is determined by the rate of proteolysis via the ubiquitin pathway. Aberrations in ubiquitin-dependent degradation of regulatory proteins frequently occur in human cancers including malignant melanoma. Melanoma cells that re-program ubiquitin-dependent proteolysis toward accelerated degradation of protein regulators of tumor suppression and abnormal stabilization of oncogenic proteins are likely to gain an advantage in growth and survival. Specific characteristics of melanoma biology include rapid metastasizing and resistance to conventional anticancer therapy. Exploration of these traits should place an emphasis on a subset of the signal transduction pathways that are governed by a number of key protein regulators whose stability and activity becomes deregulated during progression of malignant melanoma. Targeting the ubiquitination and degradation of these pivotal proteins may provide a promising new therapeutic approach to treatment of this disease.

While melanomas are resistant to the cytotoxic effects of radiotherapy, little is known about the molecular mechanisms underlying this intrinsic resistance. Here, we describe the utilization of retroviral insertional mutagenesis to facilitate the analysis of genetic changes that are associated with radioresistance in human melanoma. A radial growth phase human melanoma cell line, WM35, was infected with a replication-defective amphotropic murine retrovirus and subsequently selected for X-ray radiation-resistant variants. Several radiation-resistant clones were independently isolated and characterized. Interestingly, these clones also displayed resistance to ultraviolet radiation and to the chemotherapeutic drug cis-diamminedichloroplatinum(II) (CDDP). By Northern and Western analyses, we showed that the expression of DOPAchrome tautomerase (DCT), also known as tyrosinase-related protein 2 (TYRP2), an enzyme that functions in eumelanin synthesis, was significantly elevated in the radiation-resistant clones relative to the parental WM35 cells. Moreover, the levels of DCT in a variety of human melanoma cell lines correlated with their relative levels of radioresistance and the enforced expression of DCT conferred increased resistance to UV(B) treatment. An analysis of stress signaling induced by radiation as well other cytotoxic stressors showed that resistance associated with DCT overexpression applied specifically to treatments that activate the ERK/MAPK pathway. Indeed, DCT overexpression in a melanoma cell line resulted in increased ERK activity. Moreover, ectopic expression of dominant-active MEK in this melanoma cell line conferred UV(B) resistance suggesting that the ERK/MAPK pathway downstream of DCT may play a critical role in radiation and drug resistance. Overall, given that each gamma- and UV(B)-resistant cell line also exhibited resistance to CDDP and that CDDP-resistant clones showed increased resistance to UV(B) irradiation, these results suggest a common mechanism underlying radio- and chemoresistance, which is mediated by DCT expression.

Homologue of Slimb (HOS)/beta-transducin repeats containing proteins up-regulate nuclear factor kappaB activity by targeting its inhibitor (IkappaB) for ubiquitination and subsequent degradation. We investigated whether inhibition of HOS function may modulate apoptosis in human melanoma cells. Forced expression of the dominant negative HOSdeltaF construct inhibited IkappaB degradation and led to sensitization of melanoma cells to apoptosis induced by tumor necrosis factor alpha with cycloheximide, as well as by cisplatin and ionizing and UV irradiation. These data indicate that HOS plays an important role in controlling the IkappaB-dependent apoptotic pathways in human melanoma.

The activity of phosphatidylinositol 3-kinase (PI3K), a key component of multiple signal transduction pathways, was investigated in early- and late-stage melanoma cells that have varying degrees of radiation resistance. Analysis of PI3K biproducts (PI-3,4-P2 and PI-3,4,5-triphosphate) revealed a direct correlation between radiation resistance and levels of PI3K activity. Treating melanoma cells with wortmanin or LY294002, two different PI3K inhibitors, decreased PI3K activity and caused a dose-dependent decrease in resistance to ultraviolet radiation. Lower resistance to radiation elicited by LY294002 coincided with increased apoptosis. To further establish the role of PI3K in radiation resistance, we transfected early-stage melanoma cells with the cDNA of p85, the regulatory subunit of PI3K. Clones that constitutively overexpressed p85 exhibited a higher degree of PI-3,4-P2 synthesis and a corresponding increase in their resistance to ultraviolet radiation. The results of this study point to the role of PI3K and its biproducts in radiation resistance of human melanoma cells.

We have previously identified a U.V.-response element (URE; TGACAACA) and its bound proteins, members of the AP1 and ATF transcription factor families, in melanoma cells. Using a mutant form of cylic AMP response element binding (CREB), we found that CREB-associated-URE-bound proteins conferred characteristic melanoma phenotypes, including radiation resistance (Oncogene 12: 2223, 1996). In the present study we sought to determine which of the CREB-associated proteins confers radiation resistance on human melanoma cells. To this end we purified and identified via microsequencing ATF2 as a major URE- bound and CREB-associated protein in MeWo cells--a late stage human melanoma cell line. To determine the contribution of ATF2 to radiation resistance, MeWo cells were transfected with ATF2 cDNA lacking the trans-activation domain (ATF2(delta1-195)). MeWo cells that stably express ATF2(delta1-195) showed weaker transcriptional activities and an altered pattern of homo/hetero dimers. ATF2(delta1-195) clones exhibited up to tenfold lower resistance to irradiation by either U.V. or X-rays. The degree of resistance to radiation in the ATF2(delta1-195)-expressing clones could be increased upon transient transfection with ATF2(wt), but not with phosphorylation-defective mutant ATF2(69,71). Similarly, transfection of ATF2(wt) to WM3211, an early stage human melanoma cells line, increased resistance to radiation. Finally, changes elicited through ATF2(delta1-195) also led to reduced drug resistance, as shown for MMC, araC and cisplatinum. Our results suggest that ATF2 is a regulator of radiation and drug resistance in melanomas, and that tumor targeted ATF2 modulators may be useful sensitizers in the treatment of tumors of this type.

jun-NH2-terminal kinase (JNK) belongs to a family of protein kinases that phosphorylates c-Jun, ATF2, and Elk1 in response to various forms of stress including UV irradiation and heat shock. Although in previous studies we have demonstrated the importance of membrane components for JNK activation by UV irradiation, here we have elucidated the role of DNA damage in this response. We show that in vitro-irradiated or sonicated DNA that is added to proteins prepared from UV-treated cells can further induce JNK activation in a dose-dependent manner. When compared with UV-B (300 nm), UV-C (254 nm), which is better absorbed by the DNA, is significantly more potent in activating JNK. Furthermore, when wavelengths lower than 300 nm were filtered out, UV-B was no longer able to activate JNK. With the aid of melanoma and fibroblast cells, which exhibit different resistances to irradiation and require different UV doses to generate the same number of DNA lesions, we demonstrate that above a threshold level of 0.45 lesions and up to 0.75 lesions per 1875 bp, the degree of JNK activation correlates with the amount of lesions induced by UV-C irradiation. Finally, to explore the role of nuclear and mitochondrial DNA (mtDNA) in mediating JNK activation after UV irradiation, we have used cells that lacks mtDNA. Although the lack of mtDNA did not impair the ability of UV to activate JNK, when enucleated, these cells had lost the ability to activate JNK in response to UV irradiation. Overall, our results suggest that DNA damage in the nuclear compartment is an essential component that acts in concert with membrane-anchored proteins to mediate c-Jun phosphorylation by JNK.