Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

The Histone Chaperones ASF1 and CAF-1 Promote MMS22L-TONSL-Mediated Rad51 Loading onto ssDNA during Homologous Recombination in Human Cells.

The access-repair-restore model for the role of chromatin in DNA repair infers that chromatin is a mere obstacle to DNA repair. However, here we show that blocking chromatin assembly, via knockdown of the histone chaperones ASF1 or CAF-1 or a mutation that prevents ASF1A binding to histones, hinders Rad51 loading onto ssDNA during homologous recombination. This is a consequence of reduced recruitment of the Rad51 loader MMS22L-TONSL to ssDNA, resulting in persistent RPA foci, extensive DNA end resection, persistent activation of the ATR-Chk1 pathway, and cell cycle arrest. In agreement, histones occupy ssDNA during DNA repair in yeast. We also uncovered DNA-PKcs-dependent DNA damage-induced ASF1A phosphorylation, which enhances chromatin assembly, promoting MMS22L-TONSL recruitment and, hence, Rad51 loading. We propose that transient assembly of newly synthesized histones onto ssDNA serves to recruit MMS22L-TONSL to efficiently form the Rad51 nucleofilament for strand invasion, suggesting an active role of chromatin assembly in homologous recombination.

Lipid droplets are a metabolic vulnerability in melanoma.

Melanoma exhibits numerous transcriptional cell states including neural crest-like cells as well as pigmented melanocytic cells. How these different cell states relate to distinct tumorigenic phenotypes remains unclear. Here, we use a zebrafish melanoma model to identify a transcriptional program linking the melanocytic cell state to a dependence on lipid droplets, the specialized organelle responsible for lipid storage. Single-cell RNA-sequencing of these tumors show a concordance between genes regulating pigmentation and those involved in lipid and oxidative metabolism. This state is conserved across human melanoma cell lines and patient tumors. This melanocytic state demonstrates increased fatty acid uptake, an increased number of lipid droplets, and dependence upon fatty acid oxidative metabolism. Genetic and pharmacologic suppression of lipid droplet production is sufficient to disrupt cell cycle progression and slow melanoma growth in vivo. Because the melanocytic cell state is linked to poor outcomes in patients, these data indicate a metabolic vulnerability in melanoma that depends on the lipid droplet organelle.

Identifying the Transcriptional Drivers of Metastasis Embedded within Localized Melanoma.

In melanoma, predicting which tumors will ultimately metastasize guides treatment decisions. Transcriptional signatures of primary tumors have been utilized to predict metastasis, but which among these are driver or passenger events remains unclear. We used data from the adjuvant AVAST-M trial to identify a predictive gene signature in localized tumors that ultimately metastasized. Using a zebrafish model of primary melanoma, we interrogated the top genes from the AVAST-M signature in vivo. This identified GRAMD1B, a cholesterol transfer protein, as a bona fide metastasis suppressor, with a majority of knockout animals rapidly developing metastasis. Mechanistically, excess free cholesterol or its metabolite 27-hydroxycholesterol promotes invasiveness via activation of an AP-1 program, which is associated with increased metastasis in humans. Our data demonstrate that the transcriptional seeds of metastasis are embedded within localized tumors, suggesting that early targeting of these programs can be used to prevent metastatic relapse. SIGNIFICANCE: We analyzed human melanoma transcriptomics data to identify a gene signature predictive of metastasis. To rapidly test clinical signatures, we built a genetic metastasis platform in adult zebrafish and identified GRAMD1B as a suppressor of melanoma metastasis. GRAMD1B-associated cholesterol overload activates an AP-1 program to promote melanoma invasion. This article is highlighted in the In This Issue feature, p. 1.

Calcium-induced cleavage of DNA topoisomerase I involves the cytoplasmic-nuclear shuttling of calpain 2.

Important to the function of calpains is temporal and spatial regulation of their proteolytic activity. Here, we demonstrate that cytoplasm-resident calpain 2 cleaves human nuclear topoisomerase I (hTOP1) via Ca(2+)-activated proteolysis and nucleoplasmic shuttling of proteases. This proteolysis of hTOP1 was induced by either ionomycin-caused Ca(2+) influx or addition of Ca(2+) in cellular extracts. Ca(2+) failed to induce hTOP1 proteolysis in calpain 2-knockdown cells. Moreover, calpain 2 cleaved hTOP1 in vitro. Furthermore, calpain 2 entered the nucleus upon Ca(2+) influx, and calpastatin interfered with this process. Calpain 2 cleavage sites were mapped at K(158) and K(183) of hTOP1. Calpain 2-truncated hTOP1 exhibited greater relaxation activity but remained able to interact with nucleolin and to form cleavable complexes. Interestingly, calpain 2 appears to be involved in ionomycin-induced protection from camptothecin-induced cytotoxicity. Thus, our data suggest that nucleocytoplasmic shuttling may serve as a novel type of regulation for calpain 2-mediated nuclear proteolysis.

The Prognostic Value of Human Papilloma Virus Infection in Oral Cavity Squamous Cell Carcinoma: A Meta-Analysis.

OBJECTIVES/HYPOTHESIS: Human papilloma virus (HPV) infection has been confirmed as a favorable prognostic factor in oropharyngeal cancer. However, the prognostic value of HPV in oral squamous cell carcinoma (OSCC) is still unclear. Therefore, a systematic review and meta-analysis was performed to evaluate the prognostic value of HPV infection in OSCC patients. STUDY DESIGN: Systematic literature review with meta-analysis. METHODS: A systematic literature review was conducted in accordance with PRISMA guidelines in PubMed, EMBASE, and MEDLINE databases. The primary outcomes were overall survival (OS), disease-free survival (DFS), disease-specific survival (DSS), and secondary outcomes were local control (LC), regional control (RC), and distant control (DC). RESULTS: A total of 22 articles with 3065 OSCC patients were included in this study. Meta-analysis demonstrated that compared to HPV-negative OSCC patients, HPV-positive OSCC patients had a significantly shorter OS (hazard ratio [HR] = 1.45, 95% confidence intervals [CI], 1.10-1.93) and lower DC (HR = 2.16, 95% CI, 1.54-3.04). There was no significant difference in DFS (HR = 1.20, 95% CI, 0.63-2.26), DSS (HR = 1.20, 95% CI, 0.63-2.26), LC (HR = 1.44, 95% CI, 0.97-2.14), and RC (HR = 1.50, 95% CI, 0.98-2.30) between HPV-positive and negative OSCC patients. Sensitivity analysis confirmed the above results. CONCLUSIONS: Our systematic review and meta-analysis reveal that HPV-positive is associated with significantly decreased OS and DC, suggesting HPV infection is an adverse prognostic factor in OSCC. Laryngoscope, 132:1760-1770, 2022.

Cooperation between melanoma cell states promotes metastasis through heterotypic cluster formation.

Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.

Developmental chromatin programs determine oncogenic competence in melanoma.

Oncogenes only transform cells under certain cellular contexts, a phenomenon called oncogenic competence. Using a combination of a human pluripotent stem cell­derived cancer model along with zebrafish transgenesis, we demonstrate that the transforming ability of BRAFV600E along with additional mutations depends on the intrinsic transcriptional program present in the cell of origin. In both systems, melanocytes are less responsive to mutations, whereas both neural crest and melanoblast populations are readily transformed. Profiling reveals that progenitors have higher expression of chromatin-modifying enzymes such as ATAD2, a melanoma competence factor that forms a complex with SOX10 and allows for expression of downstream oncogenic and neural crest programs. These data suggest that oncogenic competence is mediated by regulation of developmental chromatin factors, which then allow for proper response to those oncogenes.

Cellular processing pathways contribute to the activation of etoposide-induced DNA damage responses.

Cytotoxic action (tumor cell killing) and carcinogenic side effect (therapy-related secondary leukemia) of etoposide are closely related to its ability in stabilizing topoisomerase II cleavable complex (TOP2cc), a unique form of protein-linked DNA break. How cells process and detect TOP2-concealed DNA damage for the activation of downstream cellular responses remains unclear. Here, we showed proteasomal degradation of both TOP2 isozymes in a transcription-dependent manner upon etoposide treatment. Downregulation of TOP2 was preferentially associated with proteasomal removal of TOP2 in TOP2cc rather than proteolysis of free TOP2. Interestingly, blockage of TOP2 downregulation in TOP2cc also caused reduction in etoposide-induced activation of DNA damage molecules, an observation suggesting that the processing pathways of TOP2cc are involved in activation of etoposide-induced cellular responses. In this regard, we observed two TOP2cc processing pathways, replication- and transcription-initiated processing (RIP and TIP) with proteasome involved in the latter. Importantly, two processing pathways contributed to differential activation of various DNA damage signaling and downstream cellular responses. Etoposide-induced phosphorylation of p53 relied mainly on RIP, whereas activation of Chk1, Chk2 depended largely on TIP. Both RIP and TIP played roles in activating non-homologous end joining pathway, while only RIP modulated etoposide-induced cell killing in a p53-dependent manner. Collectively, our results are consistent with the notion that protein-linked DNA breakage (e.g., TOP2cc) requires processing pathways for initiating downstream DNA damage detection, repair as well as cell death programs.

Lymph node ratio as prognostic variable in oral squamous cell carcinomas: Systematic review and meta-analysis.

Lymph node ratio (LNR) has been shown to be an independent prognostic factor for oral squamous cell carcinoma (OSCC) in various centre-based studies recently. A range of cut-off values have been suggested. A meta-analysis was performed to evaluate the prognostic effects of LNR and to investigate the cut-off value. Electronic search on Pubmed, Embase and Cochrane library and manual search were performed for studies up to January 2018. The outcomes were overall survival (OS), disease specific survival (DSS), disease free survival (DFS), local recurrence free survival (LF), locoregional disease free survival (LRF), and distant metastasis disease free survival (DM). 19 studies between 2009 and 2017 were included. The total number of patients was 14,254 (range 19-3958). Data was grouped into Group A (with pathological nodal disease, pN+) and Group B (with and without pathological nodal disease, pN+ and pN-). In the meta-analysis, the high LNR was significantly related to short OS (A = HR 1.902; 95%CI: 1.453-2.488, B = HR 2.76; 95%CI: 2.13-3.59), DSS (A = HR 1.728; 95%CI: 1.159-2.579; B = HR 2.83; 95%CI: 1.8-4.44) and DFS (A = HR 2.27; 95%CI: 1.74-2.96; B = HR 2.01; 95%CI: 1.44-2.82) in both groups; and shorter LRF in Group B (HR 5.013; 95%CI: 3.584-7.011). In the analysis, all cut-off values were shown to be significant and there was no strong evidence to consider a possibility of a second significant value. Based on our results, LNR is an independent prognostic factor in OSCC and may be considered in future oncologic staging systems.

The histone chaperone ASF1 regulates the activation of ATM and DNA-PKcs in response to DNA double-strand breaks.

The Ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are activated by DNA double-strand breaks (DSBs). These DSBs occur in the context of chromatin but how chromatin influences the activation of these kinases is not known. Here we show that loss of the replication-dependent chromatin assembly factors ASF1A/B or CAF-1 compromises ATM activation, while augmenting DNA-PKcs activation, in response to DNA DSBs. Cells deficient in ASF1A/B or CAF-1 exhibit reduced histone H4 lysine 16 acetylation (H4K16ac), a histone mark known to promote ATM activation. ASF1A interacts with the histone acetyl transferase, hMOF that mediates H4K16ac. ASF1A depletion leads to increased recruitment of DNA-PKcs to DSBs. We propose normal chromatin assembly and H4K16ac during DNA replication is required to regulate ATM and DNA-PKcs activity in response to the subsequent induction of DNA DSBs.

The Commercial Antibodies Widely Used to Measure H3 K56 Acetylation Are Non-Specific in Human and Drosophila Cells.

Much of our understanding of the function of histone post-translational modifications in metazoans is inferred from their genomic localization and / or extrapolated from yeast studies. For example, acetylation of histone H3 lysine 56 (H3 K56Ac) is assumed to be important for transcriptional regulation in metazoan cells based on its occurrence at promoters and its function in yeast. Here we directly assess the function of H3 K56Ac during chromatin disassembly from gene regulatory regions during transcriptional induction in human cells by using mutations that either mimic or prevent H3 K56Ac. Although there is rapid histone H3 disassembly during induction of some estrogen receptor responsive genes, depletion of the histone chaperone ASF1A/B, which is required for H3 K56 acetylation, has no effect on chromatin disassembly at these regions. During the course of this work, we found that all the commercially available antibodies to H3 K56Ac are non-specific in human cells and in Drosophila. We used H3-YFP fusions to show that the H3 K56Q mutation can promote chromatin disassembly from regulatory regions of some estrogen responsive genes in the context of transcriptional induction. However, neither the H3 K56R nor K56Q mutation significantly altered chromatin disassembly dynamics by FRAP analysis. These results indicate that unlike the situation in yeast, human cells do not use H3 K56Ac to promote chromatin disassembly from regulatory regions or from the genome in general. Furthermore, our work highlights the need for rigorous characterization of the specificity of antibodies to histone post-translational modifications in vivo.

The BRCA1-interacting protein Abraxas is required for genomic stability and tumor suppression.

Germline mutations of BRCA1 confer hereditary susceptibility to breast and ovarian cancer. However, somatic mutation of BRCA1 is infrequent in sporadic breast cancers. The BRCA1 protein C terminus (BRCT) domains interact with multiple proteins and are required for BRCA1's tumor-suppressor function. In this study, we demonstrated that Abraxas, a BRCA1 BRCT domain-interacting protein, plays a role in tumor suppression. Abraxas exerts its function through binding to BRCA1 to regulate DNA repair and maintain genome stability. Both homozygous and heterozygous Abraxas knockout mice exhibited decreased survival and increased tumor incidence. The gene encoding Abraxas suffers from gene copy loss and somatic mutations in multiple human cancers including breast, ovarian, and endometrial cancers, suggesting that mutation and loss of function of Abraxas may contribute to tumor development in human patients.

Sodium salicylate acts through direct inhibition of phosphoinositide 3-kinase-like kinases to modulate topoisomerase-mediated DNA damage responses.

Chemopreventive non-steroidal anti-inflammatory drugs (NSAIDs) exhibit diverse pharmacological and biological activities mainly through their inhibitory effect on cyclooxygenase (COX). However, COX-independent mechanisms involving kinase inhibition have been proposed to explain certain therapeutic effects of NSAIDs. Here, we explored the potential relationship between chemopreventive NSAIDs and DNA damage responses induced by treatment with topoisomerase-targeting drugs. (1) Sodium salicylate, a non-COX-selective NSAID, was shown to reduce DNA damage-induced RPA and p53 phosphorylation. (2) The formation of enzyme cleavable complexes by topoisomerase-targeting drugs was not affected in the presence of sodium salicylate. (3) The attenuating effect of NSAIDs on the DNA damage responses is COX-2-independent, since COX-2-selective inhibitors failed to inhibit DNA damage-induced phosphorylation of replication protein A (RPA) and p53. (4) This COX-2-independent attenuating effect was mediated through interference of neither nuclear factor kappa B nor extracellular signal-regulated kinase pathways. (5) The activation of ataxia telangiectasia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK), two key signal transducers upstream of RPA and p53, was found to be significantly reduced with sodium salicylate treatment. (6) Most importantly, sodium salicylate and other NSAIDs directly inhibited kinase activity of ATM and DNA-PK. The extent of inhibition on the kinase activity also correlated with the degree of attenuation on the DNA damage responses. (7) Unexpectedly, sodium salicylate showed a p53-independent protection effect on topoisomerase-mediated cell killing. Together, our study provides evidence that NSAIDs exhibit a novel COX-independent modulating activity of NSAIDs on the DNA damage responses and it is through inhibition of phosphoinositide 3-kinase-like kinases.

Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA breakage.

Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin (CPT) induced formation of topoisomerase I cleavable complex (TOP1cc), degradation of TOP1 and activation of DNA damage responses (DDR). Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPT-induced TOP1 degradation, while none of above three processing activities affected TOP1cc formation. Replication- and transcription-initiated processing (RIP and TIP) of TOP1cc were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOP1cc triggered the CPT-induced RPA phosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chk1/2 phosphorylation. The TIP pathway was further demonstrated to be S-phase independent by using three nonreplicating cell models. Furthermore, the effect of proteasome inhibitors mimicked that of transcription inhibition on the CPT-induced activation of DDR, suggesting the involvement of proteasome in the TIP pathway. Interestingly, the TIP pathway was important for TOP1cc-activated, but not ionization radiation-activated ATM, p53 and Chk2 phosphorylation. We have also found that pharmacological interferences of TIP and RIP pathways distinctively modulated the CPT-induced cell killing with treatments at low and high dosages, respectively. Together, our results support that both RIP and TIP pathways of TOP1cc are required for the activation of CPT-induced DDR and cytotoxicity.