Molecular Cancer Research

The pseudokinase Tribbles Homolog 1 (TRIB1) is a known driver of tumorigenesis in acute myeloid leukemia and is encoded upstream of the oncogene MYC at the 8q24 locus. We observed that TRIB1/MYC co-amplification is associated with decreased relapse-free and overall survival in breast cancer patients, but the role of TRIB1 in this disease has not been well characterized. TRIB1 knockdown in multiple breast cancer cell lines inhibited cell proliferation and suppressed MYC expression, implicating TRIB1 in breast cancer cell proliferation. Transcriptomic and cell cycle analysis revealed cell cycle regulation as the likely mechanism through which TRIB1 influences breast cancer cell proliferation. TRIB1 knockdown also resulted in significant changes in both estrogen receptor (ER) and {beta}-catenin associated transcription. Interrogating the TRIB1 interactome in breast cancer cells by qPLEX-RIME reinforced the known association between TRIB1 and ubiquitination, while revealing a range of previously undescribed TRIB1 associated factors. Further analysis of the association between TRIB1, {beta}-catenin and FERMT2 suggests TRIB1 may regulate {beta}-catenin activity by controlling the levels of both {beta}-catenin, and its co-factor FERMT2. Together, these results suggest that coregulation of {beta}-catenin and ER-driven transcription by TRIB1, facilitates regulation of MYC expression and breast cancer cell proliferation. SignificanceThe pseudokinase TRIB1 is frequently co-amplified in breast cancers with the potent oncogene MYC, although the functional consequences of this event are not well understood. This study demonstrates TRIB1 is a regulator of cell cycle progression and MYC expression in breast cancer cells. It also profiles TRIB1-associated proteins in breast cancer cells, demonstrating conservation of TRIB1s canonical interaction with COP1 and reveals associations with members of the wider ubiquitination machinery, a range of transcriptional regulators and chromatin remodelers. The data presented provide insight into the function of TRIB1 in breast cancer and the role of TRIB1 in transcriptional regulation.

Ewing sarcoma is the second most common bone cancer in children and young adults. In 85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWSR1::FLI1. EWSR1::FLI1 is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWSR1 portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWSR1::FLI1. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI1, DBD-4 helix, is critical for the transcription function of EWSR1::FLI1. In this study, we aimed to understand the mechanism by which the DBD-4 helix promotes transcription, and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatin marks, genome binding, and gene expression in cells expressing EWSR1::FLI1 constructs with and without the DBD-4 helix. Our studies revealed DBD-4 helix is crucial for cooperative binding of EWSR1::FLI1 at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWSR1::FLI1 such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.

Ewing sarcoma, an oncofusion-driven primary bone tumor, can occur in the setting of various germline mutations in DNA damage repair pathway genes. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. PARP inhibitors (PARPi) are effective against Ewing sarcoma cells in vitro, though have demonstrated limited success in clinical trials to date. In order to assess the impact of BARD1 loss on Ewing sarcoma sensitivity to PARP inhibitor therapy, we generated the novel PSaRC318 patient-derived Ewing tumor cell from our patient with a germline BARD1 mutation and then analyzed the response of these cells to PARPi. We demonstrate that PSaRC318 cells are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that loss of BARD1 enhances PARPi sensitivity. In certain malignancies, DNA damage can activate the IRF1 (interferon response factor 1) immunoregulatory pathway, and the activation of this pathway can drive immunosuppression through upregulation of the immune checkpoint protein PD-L1. In order to determine the ability of PARPi to alter Ewing tumor immunoregulation, we evaluated whether PARPi results in upregulation of the IRF1-PDL1 pathway. Indeed, we now demonstrate that PARPi leads to increased PD-L1 expression in Ewing sarcoma. Together, these data thus far suggest that while Ewing tumors harboring germline mutations in DNA damage repair proteins may in respond to PARPi in vitro, in vivo benefit of PARPi may only be demonstrated when counteracting the immunosuppressive effects of DNA damage by concurrently targeting immune checkpoint proteins.

Nuclear speckles are membrane-less bodies within the cell nucleus enriched in RNA biogenesis, processing, and export factors. In this study we investigated speckle phenotype variation in human cancer, finding a reproducible speckle signature, based on RNA expression of speckle-resident proteins, across >20 cancer types. Of these, clear cell renal cell carcinoma (ccRCC) exhibited a clear correlation between the presence of this speckle expression signature, imaging-based speckle phenotype, and clinical outcomes. ccRCC is typified by hyperactivation of the HIF-2 transcription factor, and we demonstrate here that HIF-2 drives physical association of a select subset of its target genes with nuclear speckles. Disruption of HIF-2-driven speckle association via deletion of its speckle targeting motifs (STMs)--defined in this study--led to defective induction of speckle-associating HIF-2 target genes without impacting non-speckle-associating HIF-2 target genes. We further identify the RNA export complex, TREX, as being specifically altered in speckle signature, and knockdown of key TREX component, ALYREF, also compromises speckle-associated gene expression. By integrating tissue culture functional studies with tumor genomic and imaging analysis, we show that HIF-2 gene regulatory programs are impacted by specific manipulation of speckle phenotype and by abrogation of speckle targeting abilities of HIF-2. These findings suggest that, in ccRCC, a key biological function of nuclear speckles is to modulate expression of a specific subset of HIF-2-regulated target genes that, in turn, influence patient outcomes. We also identify STMs in other transcription factors, suggesting that DNA-speckle targeting may be a general mechanism of gene regulation. HIGHLIGHTS- Nuclear speckles shown to reproducibly vary in cancer, predicting patient survival in ccRCC - HIF-2 drives DNA/gene-speckle contacts dependent on identified speckle targeting motifs within HIF-2 - Putative speckle targeting motifs are highly enriched among regulators of gene expression - Partitioning of transcription factor functional programs may be a major biological function of nuclear speckles

Chromatin remodeling plays an essential role in regulating transcriptional networks and timing of gene expression. Chromatin remodelers such as SWItch/Sucrose Non-Fermentable (SWI/SNF) harbor many protein components, with the catalytic subunit providing ATPase activity to displace histones along or from the DNA molecules, and associated subunits ensuring tissue specificity and transcriptional or co-transcriptional activities. Mutations in several of the SWI/SNF subunits have been linked to cancer. Here, we describe how SMARCD3/Baf60c expression is associated with hormone positive (ER+) breast cancer. The level SMARCD3, as detected by immunohistochemistry in breast cancer patient samples, is correlated with differential long-term disease-free survival. In contrast, the expression level of SMARCD1/Baf60a and SMARCD2/Baf60b, which are mutually exclusive within the SWI/SNF complex and have a partially redundant function, lacks predictive value in breast cancer patient samples. Lower proliferation rates are observed in SMARCD3 depleted cells, which reflects a failure to fully progress through G2/M, and an increase in endoreplication. In the absence of SMARCD3, p21 accumulates in cells but does not halt the cell cycle, and DNA damage accumulates and remains unrepaired. Taken together, our data begin to explain why ER+ breast cancer patients with low SMARCD3 expressing tumors exhibit reduced survival rates compared to patients expressing normal or higher levels of SMARCD3. SMARCD3 might act as a tumor suppressor role through regulation of cell cycle checkpoints and could be a reliable and specific breast cancer prognostic biomarker.\n\nSignificanceMutations in chromatin remodelers are a leading cause of cancer. Estrogen Receptor positive (ER+) breast cancers represent approximately 80% of all cases diagnosed. Although these tumors can be treated with hormone therapy, most breast cancer fatalities occur in ER+ breast cancer patients, due to metastasis. Low expression of SMARCD3 in ER+ cancer is associated with diminished survival rates. As such, SMARCD3 could be used as a predictive biomarker for survival. In addition, we have identified a role for SMARCD3 in the cell cycle, which could at least partially explain its protective role in breast cancer. While catalytic subunits are often viewed as the major components in chromatin remodeling function, we show here new evidence that mutations or silencing of SMARCD3 may also contribute to genomic instability and thus development of breast cancer.

HOXA9 is commonly upregulated in acute myeloid leukemia (AML), where it confers poor prognosis. Characterising the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein-SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro and prolonged survival in vivo. Integrated genomic, transcriptomic and proteomic analyses further demonstrated that the HOXA9-SAFB-chromatin complex associates with NuRD and HP1{gamma} to repress the expression of factors associated with differentiation and apoptosis, including NOTCH, CEBP{delta}, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1{gamma} catalytic activity also triggered differentiation, apoptosis and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML, that may be amenable to therapeutic intervention via therapies targeting the HOXA9/SAFB interface and/or NuRD and HP1{gamma} activity. Key Points- Identification of the endogenous human HOXA9 protein interactome in AML - HOXA9 forms a repressive complex with S/MAR binding protein (SAFB) that is critical for the maintenance of AML by facilitating proliferation and preventing differentiation and cell death. - The HOXA9/SAFB (H9SB) complex represses gene expression via recruitment of NuRD and HP1{gamma}.

ARID1B, a key subunit of the SWI/SNF (also known as BAF) chromatin remodeling complex, is characterized as a canonical tumor suppressor across various cancer types. Although the downregulation of ARID1B transcript levels has been observed in many cancers, the regulation of ARID1B at the protein level is comparatively less studied. Here, we identify WWP2, an E3 ubiquitin ligase, as a novel interacting partner of ARID1B. We further show that using its WW domains, WWP2 interacts with the PPxY motif within the N-terminal intrinsically disordered region of ARID1B. The ability of wild-type (but not the catalytically inactive) WWP2 to modulate ARID1B protein stability was confirmed through cycloheximide chase assay. Interestingly, WWP2 appears to facilitate non-canonical K27- and K29-linked polyubiquitination of ARID1B, leading to the latters proteasomal degradation. Additionally, silencing WWP2 expression results in a decrease in ubiquitination and a subsequent increase in ARID1B protein levels, indicating that WWP2 plays a crucial role in regulating ARID1B stability. Finally, based on several tumorigenic assays, we show that WWP2 may modulate ARID1B-mediated tumor suppression. Our results therefore highlight a novel mechanism of post-translational regulation of ARID1B, which may have implications in ARID1B-mediated tumor suppression. HighlightsWWP2, an E3 ubiquitin ligase, is a novel interactor of ARID1B. WWP2 regulates ARID1B protein stability by K27- and K29-linked polyubiquitination mediated proteasomal degradation. WWP2 appears to modulate the tumor suppressor activity of ARID1B by controlling its abundance in tumor cells.

Metastatic urothelial carcinoma of the bladder is generally incurable by current systemic therapy. Molecular characterization of bladder cancer (BLCa) has revealed multiple candidate driver genes for BLCa tumorigenesis. Epigenetic/chromatin modifiers have been shown to be frequently mutated in BLCa, with ARID1A mutations highly prevalent in nearly 20% of early and late stage tumors. EZH2 is a histone methyltransferase that acts as an oncogene. The data herein show that ARID1A deficient tumors, but not ARID1A wild-type tumors are sensitive to EZH2 inhibition. Specifically, EZH2 inhibitor-treated ARID1A deficient bladder cancer cells show significantly reduced cell viability, colony formation, and in vivo tumor growth relative to ARID1A-wild type bladder cancer cells. Thus, our study suggests that a specific subset of bladder cancer patients with ARID1A mutations can be therapeutically treated with pharmacologic inhibitors targeting EZH2.

The chromatin remodelers mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) subunits are mutated, deleted or amplified in more than 40% of cancers. Understanding their functions in normal cells and the consequences of cancers alterations will lead to path toward new targeted therapies. Canonically, mSWI/SNF complexes regulate the structure of chromatin, however they likely have additional functions which could be relevant in carcinogenesis. Here, we highlight the substantial alteration of mSWI/SNF subunits expression in both the nucleus and cytoplasm in breast cancer cases. We demonstrate mSWI/SNF cytoplasmic localization and interaction with the translation initiation machinery. Short-term inhibition and depletion of specific subunits alter protein synthesis, implicating a direct role for these factors in translation. Inhibition and depletion of specific subunits increase sensitivity to mTOR-PI3K inhibitors, suggesting a potential therapeutic opportunity for diseases harboring mutations in these complexes. Indeed, SMARCA4 pathogenic mutations decrease protein synthesis. Furthermore, taking advantage of the DepMap studies, we demonstrate cancer cells harboring mutations of specific mSWI/SNF subunits exhibit a genetic dependency on translation factors and are particularly sensitive to translation pathway inhibitors. In conclusion, we report an unexpected cytoplasmic role for mSWI/SNF in protein synthesis, suggesting potential new therapeutic opportunities for patients afflicted by cancers demonstrating alterations in its subunits. Statement of significanceThis study establishes direct functions for mSWI/SNF in protein synthesis. mSWI/SNF inhibition, depletion and cancer mutations alter translation and increase sensitivity to translation pathway inhibitors, illustrating the potential for new therapeutic strategies.

We recently described our initial efforts to develop a model for small cell lung cancer (SCLC) derived from human embryonic stem cells (hESCs) that were differentiated to form pulmonary neuroendocrine cells (PNECs), a putative cell of origin for neuroendocrine-positive SCLC. Although reduced expression of the tumor suppressor genes TP53 and RB1 allowed the induced PNECs to form subcutaneous growths in immune-deficient mice, the tumors did not display the aggressive characteristics of SCLC seen in human patients. Here we report that the additional, doxycycline-regulated expression of a transgene encoding wild-type or mutant cMYC protein promotes rapid growth, invasion, and metastasis of these hESC-derived cells after injection into the renal capsule. Similar to others, we find that the addition of cMYC encourages the formation of the SCLC-N subtype, marked by high levels of NEUROD1 RNA. Using paired primary and metastatic samples for RNA sequencing, we observe that the subtype of SCLC does not change upon metastatic spread and that production of NEUROD1 is maintained. We also describe histological features of these malignant, SCLC-like tumors derived from hESCs and discuss potential uses of this model in efforts to control and better understand this recalcitrant neoplasm.

Miro1 is a mitochondrial outer membrane protein that regulates mitochondrial and peroxisome trafficking, endoplasmic reticulum (ER) association, and mitophagy. Prior studies suggest a role for Miro1 in cell migration and proliferation in both normal and tumor cells. High Miro1 expression is associated with poor overall survival in breast cancer patients. To investigate the role of Miro1 in breast cancer tumorigenesis and metastasis, we established stable Miro1 knockdown (KD) in MDA-MB-231 human triple-negative breast cancer cells using shRNA. Miro1 KD significantly impaired cell proliferation, migration, and invasion in vitro. When implanted into the mammary fat pad of SCID mice, MDA-MB-231 cells formed tumors, whereas Miro1 KD cells showed markedly reduced tumorigenesis. Additionally, we generated a novel transgenic mouse model with inducible, tissue-specific Miro1 deletion in mammary epithelial cells, alongside polyomavirus middle T-antigen (PyVMT) oncogene activation. In this model, wild-type (WT) mice formed tumors at all mammary gland sites, with frequent lung metastases. However, Miro1-deficient mice failed to develop tumors, while heterozygous mice exhibited reduced tumor growth and metastasis. Additionally, these findings identify Miro1 as a key regulator of breast cancer onset and metastatic potential, positioning it as a potential biomarker and therapeutic target.

The oncogenic mechanisms by which TFE3 fusion proteins drive translocation renal cell carcinoma (tRCC) are poorly characterised. Here, we integrated loss and gain of function experiments with multi-omics analyses in tRCC cell lines and patient tumors. High nuclear accumulation of NONO-TFE3 or PRCC-TFE3 fusion proteins promotes their broad binding across the genome at H3K27ac-marked active chromatin, engaging a core set of M/E-box-containing regulatory elements to activate specific gene expression programs as well as promiscuous binding to active promoters to stimulate mRNA synthesis. Within the core program, TFE3 fusions directly regulate genes involved in ferroptosis resistance and oxidative phosphorylation metabolism (OxPhos) increasing functional OxPhos levels. Consequently, human tRCC tumors display high OxPhos scores that persist during their epithelial to mesenchymal transition (EMT). We further show that tRCC tumour aggressiveness is related to their EMT and their associated enrichment in myofibroblast cancer-associated fibroblasts (myCAFs) that are both hallmarks of poor prognostic outcomes. We define tRCC as a novel metabolic subtype of renal cancer and provide unique insights into how broad genomic binding of TFE3 fusion proteins regulates OxPhos and ferroptosis resistance and more generally stimulates RNA synthesis. SynopsisDefining the gene expression programs regulated by TFE3 fusion proteins in translocation renal cell carcinoma. TFE3 fusion proteins promote expression of oxidative metabolism genes and oxidative metabolism in tRCC. TFE3 fusion proteins regulate glutathione metabolism and ferroptosis resistance in tRCC. TFE3 fusion proteins bind broadly at active promoters and stimulate RNA synthesis in tRCC. Epithelial-to-mesenchymal transition in tRCC is accompanied by enrichment in myofibroblastic cancer-associated-fibroblasts and poor patient outcome. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/620047v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b9dcb6org.highwire.dtl.DTLVardef@495f42org.highwire.dtl.DTLVardef@10d1770org.highwire.dtl.DTLVardef@7df40c_HPS_FORMAT_FIGEXP M_FIG C_FIG The paper explained. Problem.Translocation renal cell carcinoma (tRCC) is a rare subtype of kidney cancer characterised by genetic translocation events frequently involving transcription factor TFE3 or more rarely TFEB. While the resulting fusion proteins are considered as the oncogenic drivers, their mechanism of action remains poorly understood. Results.By integrative multi-omics analyses in tRCC cell lines and patient tumors together with loss and gain of function experiments, we found broad binding of TFE3-fusion proteins at active promoters and identified a core set of target genes involved in multiple pathways, including oxidative metabolism (OxPhos) and ferroptosis. Consequently, tRCC cell lines displayed higher functional OxPhos levels and patient tumours displayed elevated OxPhos scores and ferroptosis resistance gene expression. Analyses of tRCC patient transcriptome data further revealed that mesenchymal tRCC tumours are enriched in myofibroblastic cancer associated fibroblasts that are hallmarks of poor prognostic outcome. ImpactThis study advances understanding of the molecular mechanisms underlying oncogenic transformation by TFE3 fusion proteins by defining a core program of gene expression and key features of tumour cells and their microenvironment that negatively impact patient outcome. Our integrative multi-omics and functional analyses reveal how extensive genomic binding of TFE3 fusion proteins drives high levels of oxidative metabolism, ferroptosis resistance and general RNA synthesis.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, we report that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating a role in the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. Our findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.

The Warburg effect is one of most-well studied metabolic phenomenon in cancer cells. For the most part, these studies have focused on enhanced rates of glycolysis observed in various models. The presumption has been that mitochondrial metabolism is suppressed. However, recent studies indicate that the extent of mitochondrial metabolism is far more heterogeneous in tumors than originally presumed. One tumor type with suppression of mitochondrial metabolism is renal cell carcinoma (RCC). Prior studies indicate that suppressed TCA cycle enzyme mRNA expression is associated with aggressive RCC. Yet, the mechanisms that regulate the TCA cycle in RCC remain uncharacterized. Here, we demonstrate that loss of TCA cycle enzyme expression is retained in RCC metastatic tissues. Moreover, proteomic analysis demonstrates that reduced TCA cycle enzyme expression is far more pronounced in RCC relative to other tumor types. Loss of TCA cycle enzyme expression is correlated with reduced expression of the transcription factor peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1) which is also lost in RCC tissues. PGC-1 re-expression in RCC cells restores the expression of TCA cycle enzymes in vitro and in vivo and leads to enhanced glucose carbon incorporation into TCA cycle intermediates. Mechanistically, TGF-{beta} signaling, in concert with histone deacetylase 7 (HDAC7), suppresses TCA cycle enzyme expression. In turn, pharmacologic inhibition of TGF-{beta} restores expression of TCA cycle enzyme expression and suppresses tumor growth in an orthotopic model of RCC. Taken together, our findings reveal a novel role for the TGF-{beta} /HDAC7 axis in global suppression of TCA cycle enzymes in RCC and provide novel insight into the molecular basis of altered mitochondrial metabolism in this malignancy.

Menin is a scaffolding protein that interacts with context-specific partners to regulate gene expression. In MLL-rearranged leukemias, Menin:MLL interactions drive leukemogenesis and Menin inhibitors have been FDA approved for these cancers. We previously reported that Menin promotes oncogenic phenotypes in Ewing sarcoma (EwS). Here, we sought to define EwS-specific functions of Menin and determine if Menin inhibitors could be therapeutically leveraged for these tumors. Genetic knockout of Menin had no impact on EwS cell proliferation in vitro but metastatic potential of Menin-depleted cells in vivo was impaired. Transcriptional profiling of Menin knockout cells in vitro showed reproducible downregulation of MYC signature genes and upregulation of developmental programs. Conversely, transcriptional rewiring of developmental genes and restoration of MYC target gene expression were evident in tumors that arose from Menin knockout cells. Exposing EwS cells to the Menin inhibitor VTP50469 (revumenib) inhibited expression of MYC targets and co-immunoprecipitation studies detected Menin:MYC interactions that were partially disrupted by the drug. Metastatic colonization of disseminated EwS cells in vivo was significantly inhibited in mice fed VTP50469 chow. Together these findings implicate Menin as a mediator of EwS metastasis and suggest that Menin inhibitors warrant investigation as novel therapeutics for patients with high-risk disease.

Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are frequently dysregulated in cancer. The mitotically associated lncRNA MANCR is highly expressed in aggressive cancers and contributes to genomic instability in triple-negative breast cancer (TNBC), but the molecular mechanisms underlying its activity remain poorly defined. Here we integrate computational and experimental approaches to examine the structure and regulatory interactions of MANCR isoforms. Analysis of transcriptomic datasets revealed tumor-type-specific expression patterns for seven MANCR isoforms in breast cancer cell lines. Computational prediction of RNA secondary structures identified conserved structural features across isoforms, suggesting potential functional specialization. We identify p53 as a MANCR-interacting protein through computational docking and RNA immunoprecipitation sequencing (RIP-seq) and demonstrate that MANCR depletion reduces p53-dependent transcriptional activity. Chromatin isolation by RNA purification sequencing (ChIRP-seq) revealed 1, 250 genomic regions associated with MANCR, including enrichment of p53 consensus motifs and GC-rich sequence elements. Motif analysis further identified candidate sequence features associated with MANCR-occupied chromatin regions. Computational prediction of RNA-miRNA interactions identified multiple potential miRNA binding sites across MANCR isoforms, including miR-6756-5p, which targets the androgen receptor (AR). Consistent with this prediction, AR expression decreased following MANCR knockdown in TNBC cells. Together, these results suggest that MANCR isoforms may contribute to transcriptional regulation in TNBC through interactions with chromatin, p53 signaling pathways, and potential miRNA regulatory networks. One Sentence SummaryMitotically-associated lncRNA (MANCR) is prevalent in aggressive cancers interacting with DNA, P53, and miRNAs, to mediate multiple levels of epigenetic transcriptional control in triple negative breast cancer.

High-grade complex karyotype sarcomas are a heterogeneous group of more than seventy tumors that vary in histology, clinical course, and patient demographics. Despite these clear differences, these high-grade sarcomas are treated similarly with a uniformly high metastatic rate. Pre-clinical models that allow for rigorous comparisons of distinct human sarcoma subtypes would advance insights into the relationships between sarcomas and inform therapeutic decisions. We describe the robust transformation of human mesenchymal stem cells into multiple subtypes of high-grade sarcoma. Using a pooled genetic screening approach, we identified key drivers and potential modifiers of transformation. YAP1 and KRAS were validated as drivers of two distinct sarcoma subtypes, undifferentiated pleomorphic sarcoma (UPS) and myxofibrosarcoma (MFS), respectively. In addition, the pathology of tumors driven by CDK4 and PIK3CA reflected leiomyosarcoma (LMS) and osteosarcoma (OS) indicating that further iterations of this model could result in additional sarcoma subtypes. Histologically and phenotypically these tumors reflect human sarcomas including the pathognomonic complex karyotype. In addition, CDK4 and PIK3CA driven tumors demonstrated endogenous YAP1 amplification which is seen across a subset of human tumors. While all tumors overlapped transcriptionally with the TCGA sarcoma data, further analysis confirmed that YAP1 and KRAS tumors recapitulate the UPS and MFS subtypes. Co-analysis of TCGA and model tumors support that these sarcoma subtypes lie along a spectrum of disease and adds guidance for further transcriptome-based refinement of sarcoma subtyping. Within complex karyotype sarcomas, there are multiple genetic changes but identifying those that are clinically relevant has been challenging. Comparing differentially expressed genes in YAP1 and KRAS tumors to human UPS and MFS identified the enrichment of oxidative phosphorylation pathways in both YAP1 tumors and UPS. Treatment of a panel of sarcoma cell lines with the combination of an oxidative phosphorylation inhibitor and Hippo pathway inhibitor led to a significant impairment in growth identifying new therapeutic targets. A subset of human UPS tumors showed an even greater enrichment in these pathways indicating this model can be used to identify clinically relevant subtypes. This model can be used to begin to understand pathways and mechanisms driving human sarcoma development, the relationship between sarcoma subtypes and to identify and test new therapeutic vulnerabilities for this aggressive and heterogeneous disease. Statement of SignificanceWe have created the first model to study the development, growth, and metastasis of multiple human sarcoma subtypes. This system can be used as a platform to investigate sarcoma biology and identify new therapeutic targets across a heterogeneous disease.

CD95L is expressed by tumor-infiltrating lymphocytes to eliminate CD95-expressing tumor cells and thereby CD95 loss by tumor cells is often considered as a consequence of an immunoediting process. Nonetheless CD95 expression is maintained in most triple negative breast cancers (TNBCs), and we recently reported that CD95 loss in TNBC cells triggers the induction of a pro-inflammatory program promoting the recruitment of cytotoxic NK and CD8+ T-cells and impairing tumor growth. Using a comprehensive proteomic approach, we have identified two yet unknown CD95 interaction partners, Kip1 ubiquitination-promoting complex protein 2 (KPC2) and p65. KPC2 contributes to the partial degradation of p105 (NF{kappa}B1) and the subsequent generation of p50 homodimers, which transcriptionally represses pro-inflammatory NF-{kappa}B-driven gene expression. Mechanistically, KPC2 directly interacts with the C-terminal region of CD95 and links the receptor to RelA (p65) and KPC1, the catalytic subunit of the KPC complex that acts as E3 ubiquitin-protein ligase promoting the partial degradation of p105 into p50. Loss of CD95 in TNBC cells releases KPC2, limiting the formation of the NF-{kappa}B inhibitory homodimer complex (p50/p50), promoting NF-{kappa}B activation and the production of pro-inflammatory cytokines including CSF1, CSF2, CXCL1 and IL1 members, known to promote recruitment and differentiation of certain adaptive and innate immune effector cells.

The transcription factor Activating Protein 2{beta} (TFAP2B; AP-2{beta}) is a candidate marker for the molecular apocrine subtype of breast cancer, which lacks expression of the estrogen receptor alpha (ESR1; ER) but sustains a luminal breast cancer phenotype due to the presence of key luminal lineage transcription factors (GATA3, FOXA1) and activity of the androgen receptor (AR). Our objective was to better understand the expression patterns and molecular function of AP-2{beta} in breast cancer. Using multiple clinical cohorts, we show that high TFAP2B expression was associated with low proliferation in ER positive (ER+) breast cancers of ductal and lobular histology, but with enrichment for lobular tumours. High TFAP2B was also evident in high proliferation tumours lacking ER, with no specific enrichment for lobular histology. Restoring AP-2{beta} expression to highly proliferative ER+ breast cancer cell lines that lack it strongly induced apoptosis. Conversely, reducing AP-2{beta} expression in a molecular apocrine breast cancer cell line model potently inhibited proliferation and cell viability associated with downregulation of MYC oncogene expression. To identify genomic determinants of AP-2{beta} function in molecular apocrine cells in relation to other known transcriptional regulators, we performed chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) for AP-2{beta}, AR, GATA3, FOXA1, and acetylated lysine 27 in histone 3 (H3K27ac, a marker of active enhancers and promoters). When present alone, AP-2{beta} was preferentially enriched at active promotors. Enhancers bound by AR, GATA3 and FOXA1 were more active when AP-2{beta} was present and genes that define the molecular apocrine phenotype were significantly more likely to have active enhancers co-occupied by all four transcription factors. We conclude that AP-2{beta} plays a context-dependent role in breast cancer and propose that activation of enhancers defining molecular apocrine identity is a key function of AP-2{beta} in the biology of this subtype of breast cancer.

Increased presence of myeloid derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in tumor tissue has been extensively reported. These cells represent a major constituent of tumor infiltrate and exhibit a distinct phenotype with immunosuppressive and tolerogenic functions. However, their role in the regulation of hyaluronan (HA) metabolism in the tumor microenvironment has not been established. Here we describe a novel function of tumor-associated myeloid cells related to the enhanced breakdown of extracellular HA in human bladder cancer tissue leading to accumulation of small HA fragments with MW <20 kDa. Increased fragmentation of extracellular HA and accumulation of low molecular weight HA (LMW-HA) in tumor tissue was associated with elevated production of multiple inflammatory cytokines, chemokines, and angiogenic factors. The fragmentation of HA by myeloid cells was mediated by the membrane-bound enzyme hyaluronidase 2 (Hyal2). The increased numbers of Hyal2+CD11b+ myeloid cells were detected in the tumor tissue as well as in the peripheral blood of bladder cancer patients. Co-expression of CD33 suggests that these cells belong to monocytic myeloid-derived suppressor cells. HA-degrading function of Hyal2-expressing MDSCs could be enhanced by exposure to tumor-conditioned medium, and IL-1{beta} was identified as one of factors involved in the stimulation of Hyal2 activity. CD44-mediated signaling plays an important role in the regulation of HA-degrading activity of Hyal2-expressing myeloid cells, since engagement of CD44 receptor with specific monoclonal antibody triggered translocation of Hyal2 enzyme to the cellular surface and also stimulated secretion of IL-1{beta}. Taken together, this work identifies the Hyal2-expressing tumor-associated myeloid cells, and links these cells to the accumulation of LMW-HA in the tumor microenvironment and cancer-related inflammation and angiogenesis.