CTCF binding sites are frequently mutated in cancer, but how these mutations accumulate and whether they broadly perturb CTCF binding are not well understood. Here, we report that skin cancers exhibit a highly specific asymmetric mutation pattern within CTCF motifs attributable to ultraviolet irradiation and differential nucleotide excision repair (NER). CTCF binding site mutations form independently of replication timing and are enriched at sites of CTCF/cohesin complex binding, suggesting a role for cohesin in stabilizing CTCF-DNA binding and impairing NER. Performing CTCF ChIP-seq in a melanoma cell line, we show CTCF binding site mutations to be functional by demonstrating allele-specific reduction of CTCF binding to mutant alleles. While topologically associating domains with mutated CTCF anchors in melanoma contain differentially expressed cancer-associated genes, CTCF motif mutations appear generally under neutral selection. However, the frequency and potential functional impact of such mutations in melanoma highlights the need to consider their impact on cellular phenotype in individual genomes.

BackgroundCancer cells require increased levels of nutrients such as amino acids to sustain their rapid growth. In particular, leucine and glutamine have been shown to be important for growth and proliferation of some breast cancers, and therefore targeting the primary cell-surface transporters that mediate their uptake, L-type amino acid transporter 1 (LAT1) and alanine, serine, cysteine-preferring transporter 2 (ASCT2), is a potential therapeutic strategy.MethodsThe ASCT2 inhibitor, benzylserine (BenSer), is also able to block LAT1 activity, thus inhibiting both leucine and glutamine uptake. We therefore aimed to investigate the effects of BenSer in breast cancer cell lines to determine whether combined LAT1 and ASCT2 inhibition could inhibit cell growth and proliferation.ResultsBenSer treatment significantly inhibited both leucine and glutamine uptake in MCF-7, HCC1806 and MDA-MB-231 breast cancer cells, causing decreased cell viability and cell cycle progression. These effects were not primarily leucine-mediated, as BenSer was more cytostatic than the LAT family inhibitor, BCH. Oocyte uptake assays with ectopically expressed amino acid transporters identified four additional targets of BenSer, and gas chromatography-mass spectrometry (GCMS) analysis of intracellular amino acid concentrations revealed that this BenSer-mediated inhibition of amino acid uptake was sufficient to disrupt multiple pathways of amino acid metabolism, causing reduced lactate production and activation of an amino acid response (AAR) through activating transcription factor 4 (ATF4).ConclusionsTogether these data showed that BenSer blockade inhibited breast cancer cell growth and viability through disruption of intracellular amino acid homeostasis and inhibition of downstream metabolic and growth pathways.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4599-8) contains supplementary material, which is available to authorized users.

Cancer-Associated Fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through pro-tumor signalling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDACderived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth.Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle genesilencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. Statement of Significance:This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAF and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

Loss of the retinoblastoma (RB) tumor suppressor protein is a critical step in reprogramming biological networks that drive cancer progression, although mechanistic insight has been largely limited to the impact of RB loss on cell cycle regulation. Here, isogenic modeling of RB loss identified disease stage-specific rewiring of E2F1 function, providing the first-in-field mapping of the E2F1 cistrome and transcriptome after RB loss across disease progression. Biochemical and functional assessment using both in vitro and in vivo models identified an unexpected, prominent role for E2F1 in regulation of redox metabolism after RB loss, driving an increase in the synthesis of the antioxidant, glutathione, specific to advanced disease. These E2F1-dependent events resulted in protection from reactive oxygen species (ROS) in response to therapeutic intervention. On balance, these findings reveal novel pathways through which RB loss promotes cancer progression and highlight potentially new nodes of intervention for treating RB-deficient cancers.Research.

Androgen receptor (AR) is a ligand-induced transcriptional factor, which plays an important role in the normal development of prostate as well as in the progression of prostate cancer. Numerous coactivators, which associate with AR and function to remodel chromatin and recruit RNA polymerase II to enhance the transcriptional potential of AR, have been identified. Among these coactivators, few are protein kinases. In this study, we describe the characterization of a novel protein kinase, male germ cell-associated kinase (MAK), which serves as a coactivator of AR. We present evidence, which indicates that (a) MAK physically associates with AR (MAK and AR are found to be coprecipitated from cell extracts, colocalized in nucleus, and corecruited to prostate-specific antigen promoter in LNCaP as well as in transfected cells); (b) MAK is able to enhance AR transactivation potential in an androgen-and kinase-dependent manner in several prostate cancer cells and synergize with ACTR/steroid receptor coactivator-3 coactivator; (c) small hairpin RNA (shRNA) knocks down MAK expression resulting in the reduction of AR transactivation ability; (d) MAK-shRNA or kinase-dead mutant, when introduced into LNCaP cells, reduces the growth of the cells; and (e) microarray analysis of LNCaP cells carrying kinase-dead MAK mutant showed a significant impediment of AR signaling, indicating that endogenous MAK plays a general role in AR function in prostate cancer cells and likely to be a general coactivator of AR in prostate tissues. The highly restricted expression of this kinase makes it a potentially useful target for intervention of androgen independence. (Cancer Res 2006; 66(17): 8439-47)