Cylindromatosis Is Required for Survival of a Subset of Melanoma Cells.

The deubiquitinase cylindromatosis (CYLD) functions as a tumor suppressor inhibiting cell proliferation in many cancer types including melanoma. Here we present evidence that a proportion of melanoma cells are nonetheless addicted to CYLD for survival. The expression levels of CYLD varied widely in melanoma cell lines and melanomas in vivo, with a subset of melanoma cell lines and melanomas displaying even higher levels of CYLD than melanocyte lines and nevi, respectively. Strikingly, although short hairpin RNA (shRNA) knockdown of CYLD promoted, as anticipated, cell proliferation in some melanoma cell lines, it reduced cell viability in a fraction of melanoma cell lines with relatively high levels of CYLD expression and did not impinge on survival and proliferation in a third type of melanoma cell lines. The decrease in cell viability caused by CYLD knockdown was due to induction of apoptosis, as it was associated with activation of the caspase cascade and was abolished by treatment with a general caspase inhibitor. Mechanistic investigations demonstrated that induction of apoptosis by CYLD knockdown was caused by upregulation of receptor-interacting protein kinase 1 (RIPK1) that was associated with elevated K63-linked polyubiquitination of the protein, indicating that CYLD is critical for controlling RIPK1 expression in these cells. Of note, microRNA (miR) profiling showed that miR-99b-3p that was predicted to target the 3-untranslated region (3-UTR) of the CYLD mRNA was reduced in melanoma cell lines with high levels of CYLD compared with melanocyte lines. Further functional studies confirmed that the reduction in miR-99b-3p expression was responsible for the increased expression of CYLD in a highly cell line-specific manner. Taken together, these results reveal an unexpected role of CYLD in promoting survival of a subset of melanoma cells and uncover the heterogeneity of CYLD expression and its biological significance in melanoma.

LncRNA REG1CP promotes tumorigenesis through an enhancer complex to recruit FANCJ helicase for REG3A transcription.

Protein products of the regenerating islet-derived (REG) gene family are important regulators of many cellular processes. Here we functionally characterise a non-protein coding product of the family, the long noncoding RNA (lncRNA) REG1CP that is transcribed from a DNA fragment at the family locus previously thought to be a pseudogene. REG1CP forms an RNA-DNA triplex with a homopurine stretch at the distal promoter of the REG3A gene, through which the DNA helicase FANCJ is tethered to the core promoter of REG3A where it unwinds double stranded DNA and facilitates a permissive state for glucocorticoid receptor α (GRα)-mediated REG3A transcription. As such, REG1CP promotes cancer cell proliferation and tumorigenicity and its upregulation is associated with poor outcome of patients. REG1CP is also transcriptionally inducible by GRα, indicative of feedforward regulation. These results reveal the function and regulation of REG1CP and suggest that REG1CP may constitute a target for cancer treatment.

Correction to: RIP1 protects melanoma cells from apoptosis induced by BRAF/MEK inhibitors.

Since publication of this paper, the authors have noticed that there were errors in Fig. 2A (the GAPDH of Mel-CV, Mel-CV.S, Mel-RMu and Mel-RMu.S), Fig. 2C (the GAPDH of Mel-CV.S and Mel-RMu.S), Fig. 3F (the GAPDH of Mel-CV.S and Mel-RMu.S), Fig. 3J(the GAPDH of Mel-RMu.S), Fig. 5C (the ERK1/2 of patient#3(post)), and Fig. 5F (the RIP1 of Mel-CV.S and Mel-RMu.S, the GAPDH of Mel-CV and Mel-RMu). As a result of the misfiling of the data during preparation of figures, incorrect images were inadvertently inserted in these figures. The correct figures are given below. The corrections do not alter the conclusions of the paper.

A p53-Responsive miRNA Network Promotes Cancer Cell Quiescence.

: Cancer cells in quiescence (G0 phase) are resistant to death, and re-entry of quiescent cancer cells into the cell-cycle plays an important role in cancer recurrence. Here we show that two p53-responsive miRNAs utilize distinct but complementary mechanisms to promote cancer cell quiescence by facilitating stabilization of p27. Purified quiescent B16 mouse melanoma cells expressed higher levels of miRNA-27b-3p and miRNA-455-3p relative to their proliferating counterparts. Induction of quiescence resulted in increased levels of these miRNAs in diverse types of human cancer cell lines. Inhibition of miRNA-27b-3p or miRNA-455-3p reduced, whereas its overexpression increased, the proportion of quiescent cells in the population, indicating that these miRNAs promote cancer cell quiescence. Accordingly, cancer xenografts bearing miRNA-27b-3p or miRNA-455-3p mimics were retarded in growth. miRNA-27b-3p targeted cyclin-dependent kinase regulatory subunit 1 (CKS1B), leading to reduction in p27 polyubiquitination mediated by S-phase kinase-associated protein 2 (Skp2). miRNA-455-3p targeted CDK2-associated cullin domain 1 (CAC1), which enhanced CDK2-mediated phosphorylation of p27 necessary for its polyubiquitination. Of note, the gene encoding miRNA-27b-3p was embedded in the intron of the chromosome 9 open reading frame 3 gene that was transcriptionally activated by p53. Similarly, the host gene of miRNA-455-3p, collagen alpha-1 (XXVII) chain, was also a p53 transcriptional target. Collectively, our results identify miRNA-27b-3p and miRNA-455-3p as important regulators of cancer cell quiescence in response to p53 and suggest that manipulating miRNA-27b-3p and miRNA-455-3p may constitute novel therapeutic avenues for improving outcomes of cancer treatment. SIGNIFICANCE: Two novel p53-responsive microRNAs whose distinct mechanisms of action both stabilize p27 to promote cell quiescence and may serve as therapeutic avenues for improving outcomes of cancer treatment.

RIP1 protects melanoma cells from apoptosis induced by BRAF/MEK inhibitors.

Many recent studies have uncovered the necessary role for the receptor-interacting protein kinase 1 (RIP1) in regulating apoptosis and necrosis that cells undergo in response to various cellular stresses. However, the functional significance of RIP1 in promoting cancer cells survival remains poorly understood. Here, we report that RIP1 was upregulated and contributed to both intrinsic and acquired resistance of melanoma cells to BRAF/MEK inhibitors through activation of NF-κB. Strikingly, Snail1-mediated suppression of CYLD played a crucial role in promoting RIP1 expression upon ERK activation, particularly, in melanoma cells with acquired resistance to BRAF inhibitors. In addition, RIP1 kinase activity was not required for melanoma cells to survive BRAF/MEK inhibition as RIP1 mediated NF-κB activation through its intermediate domain. Collectively, our findings reveal that targeting RIP1 in combination with BRAF/MEK inhibitors is a potential approach in the treatment of the disease.

ACTN4 regulates the stability of RIPK1 in melanoma.

The actin crosslinking protein α-actinin-4 (ACTN4) is emerging as an important contributor to the pathogenesis of cancer. This has largely been attributed to its role in regulating cytoskeleton organization and its involvement in transcriptional regulation of gene expression. Here we report a novel function of ACTN4 as a scaffold necessary for stabilization of receptor-interacting protein kinase 1 (RIPK1) that we have recently found to be an oncogenic driver in melanoma. ACTN4 bound to RIPK1 and cellular inhibitor of apoptosis protein 1 (cIAP1) with its actin-binding domain at the N-terminus and the CaM-like domain at the C-terminus, respectively. This facilitated the physical association between RIPK1 and cIAP1 and was critical for stabilization of RIPK1 that in turn activated NF-κB. Functional investigations showed that silencing of ACTN4 suppressed melanoma cell proliferation and retarded melanoma xenograft growth. In contrast, overexpression of ACTN4 promoted melanocyte and melanoma cell proliferation and moreover, prompted melanocyte anchorage-independent growth. Of note, the expression of ACTN4 was transcriptionally activated by NF-κB. Taken together, our findings identify ACTN4 as an oncogenic regulator through driving a feedforward signaling axis of ACTN4-RIPK1-NF-κB, with potential implications for targeting ACTN4 in the treatment of melanoma.

Cooperativity of HOXA5 and STAT3 Is Critical for HDAC8 Inhibition-Mediated Transcriptional Activation of PD-L1 in Human Melanoma Cells.

Although the expression of programmed death-ligand 1 (PD-L1) is an important mechanism by which cancer cells evade the immune system, PD-L1 expression in cancer cells is commonly associated with patients' responses to treatment with anti-programmed death 1/PD-L1 antibodies. However, how PD-L1 expression is regulated in melanoma cells remains to be fully elucidated. Here we report that the class I histone deacetylase (HDAC) HDAC8 controls transcriptional activation of PD-L1 by a transcription complex consisting of transcription factors homeobox A5 and signal transducer and activator of transcription 3. Inhibition of HDAC8 upregulated PD-L1 in melanoma cells. This was due to an increase in the activity of a fragment of the PD-L1 gene promoter that is enriched with binding sites for both homeobox A5 and signal transducer and activator of transcription 3. Indeed, knockdown of homeobox A5 or signal transducer and activator of transcription 3 abolished upregulation of PD-L1 by HDAC8 inhibition. Moreover, homeobox A5 and signal transducer and activator of transcription 3 were physically associated and appeared interdependent in activating PD-L1 transcription. Functional studies showed that HDAC8-mediated regulation of PD-L1 expression participated in modulating anti-melanoma T-cell responses. Collectively, these results identify HDAC8 as an important epigenetic regulator of PD-L1 expression, with implications for better understanding of the interaction between melanoma cells and the immune system.

BRAF/MEK inhibitors promote CD47 expression that is reversible by ERK inhibition in melanoma.

The expression of CD47 on the cancer cell surface transmits "don't eat me" signalling that not only inhibits phagocytosis of cancer cells by phagocytes but also impairs anti-cancer T cell responses. Here we report that oncogenic activation of ERK plays an important role in transcriptional activation of CD47 through nuclear respiratory factor 1 (NRF-1) in melanoma cells. Treatment with BRAF/MEK inhibitors upregulated CD47 in cultured melanoma cells and fresh melanoma isolates. Similarly, melanoma cells selected for resistance to the BRAF inhibitor vemurafenib expressed higher levels of CD47. The increase in CD47 expression was mediated by ERK signalling, as it was associated with rebound activation of ERK and co-knockdown of ERK1/2 by siRNA diminished upregulation of CD47 in melanoma cells after exposure to BRAF/MEK inhibitors. Furthermore, ERK1/2 knockdown also reduced the constitutive expression of CD47 in melanoma cells. We identified a DNA fragment that was enriched with the consensus binding sites for NRF-1 and was transcriptionally responsive to BRAF/MEK inhibitor treatment. Knockdown of NRF-1 inhibited the increase in CD47, indicating that NRF-1 has a critical role in transcriptional activation of CD47 by ERK signalling. Functional studies showed that melanoma cells resistant to vemurafenib were more susceptible to macrophage phagocytosis when CD47 was blocked. So these results suggest that NRF-1-mediated regulation of CD47 expression is a novel mechanism by which ERK signalling promotes the pathogenesis of melanoma, and that the combination of CD47 blockade and BRAF/MEK inhibitors may be a useful approach for improving their therapeutic efficacy.

Functional identification of a novel transcript variant of INPP4B in human colon and breast cancer cells.

The 4-phosphatase Inositol polyphosphate 4-phosphatase II (INPP4B) is a regulator of the PI3K signalling pathway and functions to suppress or promote activation of downstream kinases depending on cell type and context. Here we report the identification of a novel small transcript variant of INPP4B (INPP4B-S) that has a role in promoting proliferation of colon and breast cancer cells. INPP4B-S differed from full length INPP4B (INPP4B-FL) by the insertion of a small exon between exons 15 and 16 and the deletion of exons 20-24. Nevertheless, INPP4B-S retained all the functional domains of INPP4B-FL and was similarly located to the cytoplasm. Overexpression of INPP4B-S increased, whereas selective knockdown of INPP4B-S reduced the rate of proliferation in HCT116 and MCF-7 cells. These results warrant further investigation of the role INPP4B-S in activation of downstream kinases and in regulation of cancer pathogenesis.

Reactive Oxygen Species Dictate the Apoptotic Response of Melanoma Cells to TH588.

The effect of MTH1 inhibition on cancer cell survival has been elusive. Here we report that although silencing of MTH1 does not affect survival of melanoma cells, TH588, one of the first-in-class MTH1 inhibitors, kills melanoma cells through apoptosis independently of its inhibitory effect on MTH1. Induction of apoptosis by TH588 was not alleviated by MTH1 overexpression or introduction of the bacterial homolog of MTH1 that has 8-oxodGTPase activity but cannot be inhibited by TH588, indicating that MTH1 inhibition is not the cause of TH588-induced killing of melanoma cells. Although knockdown of MTH1 did not impinge on the viability of melanoma cells, it rendered melanoma cells sensitive to apoptosis induced by the oxidative stress inducer elesclomol. Of note, treatment with elesclomol also enhanced TH588-induced apoptosis, whereas a reactive oxygen species scavenger or an antioxidant attenuated the apoptosis triggered by TH588. Indeed, the sensitivity of melanoma cells to TH588 was correlated with endogenous levels of reactive oxygen species. Collectively, these results indicate that the cytotoxicity of TH588 toward melanoma cells is not associated with its inhibitory effect on MTH1, although it is mediated by cellular production of ROS.

Reactivation of ERK and Akt confers resistance of mutant BRAF colon cancer cells to the HSP90 inhibitor AUY922.

Oncogenic mutations of BRAF occur in approximately 10% of colon cancers and are associated with their resistance to clinically available therapeutic drugs and poor prognosis of the patients. Here we report that colon cancer cells with mutant BRAF are also resistant to the heat shock protein 90 (HSP90) inhibitor AUY922, and that this is caused by rebound activation of ERK and Akt. Although AUY922 triggered rapid reduction in ERK and Akt activation in both wild-type and mutant BRAF colon cancer cells, activation of ERK and Akt rebounded shortly in the latter leading to resistance of the cells to AUY922-induced apoptosis. Reactivation of ERK was associated with the persistent expression of mutant BRAF, which, despite being a client of HSP90, was only partially degraded by AUY922, whereas reactivation of Akt was related to the activity of the HSP90 co-chaperone, cell division cycle 37 (CDC37), in that knockdown of CDC37 inhibited Akt reactivation in mutant colon cancer cells treated with AUY922. In support, as a HSP90 client protein, Akt was only diminished by AUY922 in wild-type but not mutant BRAF colon cancer cells. Collectively, these results reveal that reactivation of ERK and Akt associated respectively with the activity of mutant BRAF and CDC37 renders mutant BRAF colon cancer cells resistant to AUY922, with implications of co-targeting mutant BRAF and/or CDC37 and HSP90 in the treatment of mutant BRAF colon cancers.

OBATOCLAX and ABT-737 induce ER stress responses in human melanoma cells that limit induction of apoptosis.

Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.

Evidence for upregulation of Bim and the splicing factor SRp55 in melanoma cells from patients treated with selective BRAF inhibitors.

Relatively little attention has been paid to the activity of selective BRAF inhibitors in the induction of apoptosis in melanoma, particularly in vivo. In the present study, we have isolated cultures from biopsies taken from four patients before and during the treatment of their melanoma. We report that the cell lines taken during treatment show varying degrees of upregulation of the proapoptotic BH3 protein Bim and its splice forms, downregulation of Mcl-1, and upregulation of the splicing factor SRp55 as reported in previous in-vitro studies. There was also evidence of ongoing apoptotic signaling despite the continued growth of the cultures. The cultures established during the treatment were largely resistant to the selective BRAF inhibitor PLX4720, consistent with the acquired resistance of melanoma in the treated patients. These results provide further insights into the mechanism of action of these agents against melanoma.

Phase I/II study of immunotherapy with T-cell peptide epitopes in patients with stage IV melanoma.

Previous studies in small groups of patients suggested that immunization of melanoma patients with peptide epitopes recognized by T cells could induce regression of melanoma. This approach was tested in 36 patients with stage IV melanoma. The (MHC class I-restricted) peptides were from gp100, MART-1, tyrosinase, and MAGE-3. The gp100 and MART-1 peptides had been modified to increase their immunogenicity. In half the patients (groups 3 and 4) the peptides were given in the adjuvant Montanide-ISA-720, and half the patients in both groups were given GM-CSF s.c. for 4 days following each injection. Treatment was well tolerated except for two severe erythematous responses to Montanide-ISA-720 and marked inflammatory responses at sites of GM-CSF administration in three patients. There were no objective clinical responses but stabilization of disease for periods from 3 to 12 months were seen in seven patients. Five of these were patients given the peptides in Montanide-ISA-720. Delayed-type hypersensitivity (DTH) skin test responses were also seen mainly in the patients given the peptides in Montanide-ISA-720. GM-CSF did not increase DTH responses in patients in the latter group but may have increased DTH responses in those not given peptides in Montanide-ISA-720. Inflammatory responses around s.c. metastases or regional lymph nodes were observed in two patients. These results suggest that the peptides are more effective when given in the adjuvant Montanide-ISA-720. Nevertheless, results from this study, together with those from a number of comparable studies, indicate that peptide vaccines are currently of minimal benefit to patients and support the need for ongoing development of new strategies in treatment of this disease.

Phase I/II study of treatment with dendritic cell vaccines in patients with disseminated melanoma.

Previous studies have suggested that immunotherapy with dendritic cell (DC) vaccines may be effective in treatment of patients with AJCC stage IV melanoma. We examined this treatment in phase I/II studies in 33 patients with good performance status and low volume disease. Nineteen patients received DCs plus autologous lysates and 14 patients DCs plus peptides from the melanoma antigens MAGE-3.A2, tyrosinase, gp100, and MART-1. Keyhole limpet hemocyanin (KLH) was used as a helper protein and influenza peptide was given as a positive control. DCs were produced from adherent cells in blood lymphocytes (monocytic DCs), grown in IL-4 and GM-CSF without a maturation step. The DCs were injected into inguinal lymph nodes at weekly intervals (x4), 2 weeks (x1), and 4-weekly intervals (x2). There were 3 responses (3 partial responses) and 1 mixed response in the 19 patients treated with DCs plus autologous lysates. No responses were seen in the group treated with DCs plus peptides. Stable disease (defined as no progression over a period of 3 months) was seen in 4 patients in group 1 and 5 patients in group 2. Treatment was not associated with significant side effects. We examined whether DTH skin tests or assays of IFN-gamma cytokine production may be useful predictors of clinical responses. Twenty-two of 30 patients had DTH responses to KLH and 12 of 13 patients had DTH responses to the influenza peptide. Five of 15 DTH responses were seen against autologous lysates. This was strongly correlated with clinical responses. Approximately half the patients had responses to MART-1 peptide and a third to the other melanoma peptides. Similarly, cytokine production assays showed responses to influenza in 7 of 13 patients, and approximately one third of patients had responses to the other peptides. No IFN-gamma responses were seen in 5 patients against their autologous lysates. There was no correlation between assays of IFN-gamma production and clinical responses. The present studies suggest that autologous lysates may be more effective than the melanoma peptides used in the study as the source of antigen for DC vaccines. DTH responses to autologous lysates appear useful predictors of clinical responses, but further work is needed to identify other measures associated with clinical responses.