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The αvβ6 integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of αvβ6 expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of αvβ6 in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent αvβ6 expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate αvβ6 expression. Detroit 562 cells showed αvβ6-dependent adhesion and activation of transforming growth factor-β (TGF-β) that was inhibited >90% with an αvβ6 blocking antibody, 6.3G9. Although both recombinant soluble TGF-β receptor type-II (rsTGF-βRII-Fc) and 6.3G9 inhibited TGF-β–mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-βRII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by αvβ6 antibodies, our findings support a role for αvβ6 in human cancer and underscore the therapeutic potential of function blocking αvβ6 antibodies. [Cancer Res 2008;68(2):561–70]

Genetic changes in epithelial cells initiate the development of prostatic adenocarcinomas. As nascent tumors grow and undergo progression, epithelial tumor cells are intimately associated with stromal cells. Stromal cells within the tumor microenvironment acquire new properties, including the capacity to promote phenotypic and genetic progression in adjacent epithelial cells. Affymetrix microarrays were used to identify 119 genes differentially expressed between normal-derived and carcinoma-derived prostatic stromal cells. These included 31 genes encoding extracellular proteins that may act as stromal-to-epithelial paracrine signals. Further investigation of one of these genes, secreted frizzled related protein 1 (SFRP1), revealed that its expression parallels prostatic growth with high expression during prostatic development, low expression in the adult prostate, and elevated expression in prostatic tumor stroma. In addition, as prostatic epithelial cells progressed to a tumorigenic state under the influence of tumor stroma, SFRP1 became overexpressed in the progressed epithelial cells. To further understand the roles of SFRP1 in the prostate, we tested the affects of increased SFRP1 levels on prostatic tissues and cells. Treatment of developing prostates with SFRP1 in culture led to increased organ growth. Treatment of a human prostatic epithelial cell line with SFRP1 led to increased proliferation, decreased apoptosis, and decreased signaling through the Wnt/β-catenin pathway in vitro and increased proliferation in vivo. These data suggest that overexpression of SFRP1 by prostatic tumor stroma may account for the previously reported capacity of prostatic tumor stroma to provide a pro-proliferative paracrine signal to adjacent epithelial cells.

The bromodomain and extra-terminal (BET) protein, BRD4 functions as a transcriptional co-activator through recruitment of regulatory complexes to acetylated chromatin. Among all malignancies, the most focal, and recurrent BRD4 gene amplification occurs in high-grade serous ovarian cancer (HGSOC). Despite the elucidation of BRD4 dependencies in cancer, it is unclear whether BRD4 gene amplification can result in oncogenic activity, and hence serve as a patient selection strategy for BET bromodomain inhibitors. To assess the oncogenic potential of BRD4 amplification in HGSOC, we assayed enforced BRD4 expression in non-transformed immortalized surface ovarian cells (IOSE). Physiologically relevant expression of either long or short BRD4 spliced isoforms in IOSE (BRD4-IOSE) resulted in marked phenotypic changes indicative of cellular transformation, including altered growth kinetics, chromatin reorganization, and acquisition of colony formation potential. Transcriptional profiling of BRD4-IOSE and BRD4-amplified HGSOC patients revealed shared expression patterns, including enriched MYC, E2F1, and VEGF gene signatures. Interrogation of BRD4 inhibitor pharmacodynamic markers, and transcripts upregulated through enforced BRD4 expression uncovered Neuregulin-1 as a critical BRD4 transcriptional effector. RNAi-mediated Neuregulin-1 depletion was capable of abolishing the oncogenic activity of BRD4-IOSE, and exogenous Neuregulin-1 was sufficient to transform IOSE without BRD4. A novel BRD4-SWI/SNF interaction was required for driving the oncogenic activity of BRD4-IOSE through maintaining NRG1 expression. The sensitivity of patient-derived HGSOC xenografts to a novel clinical candidate BRD4 inhibitor, AZD5153, correlated with BRD4 amplification and a pharmacodynamic NRG1 response. This study demonstrates the oncogenic potential of BRD4 amplification, defines a relationship between BRD4 and NRG1, and further establishes HGSOC as an appropriate patient population in which to test the therapeutic potential of BET bromodomain inhibitors. Citation Format: Garrett W. Rhyasen, Yi Yao, Austin Dulak, Lillian Castriotta, Kelly Jacques, Maureen Hattersley, Gordon B. Mills, Michael Zinda, Stephen Fawell, Paul Lyne, Edwin Clark, Huawei Chen. BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1971.

Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor α (TNFα) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself. Here, we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TNFα signaling. Mechanistic studies indicate that in this context, XIAP is a positive modulator of TNFα induction whereas cellular inhibitor of apoptosis protein 1 negatively regulates TNFα-mediated apoptosis. [Cancer Res 2007;67(24):11493–8]

Our current understanding of the mutation spectrum of relapse/refractory patients is limited. Several published studies describing the mutational landscape of Diffuse Large B cell Lymphoma (DLBCL) have focused, by design, on diagnostic (pre-treatment) biopsies alone, while re-biopsy of patients who are refractory to first line therapy or who relapse on treatment is not standard of care in DLBCL. We have performed whole exome sequencing on 47 post treatment DLBCL core needle biopsies, 8 with matched diagnostic biopsies. Samples were obtained during a phase II trial prior to the start of treatment with the BCR targeted agent fostamatinib (1,2). Patients had progressed following therapy with an anthracycline-based regimen such as R-CHOP and had a median of 3 prior therapies (range 1-8). We compared the whole exome somatic variant and copy number calls from the post treatment and matched pairs to 2 cohorts of primary DLBCL (n = 112) analyzed with the same mutation caller (3,4). The average mutation rate between the pre and post treatment samples (n = 250, 282 respectively) and the paired diagnostic and post treatment biopsies were similar. However, we found that the mutation spectrum between the paired biopsies differed. Known DLBCL hotspot mutations such as MYD88 L265P remained consistent between pre and post treatment biopsies, however novel mutations in known DLBCL targets were found to emerge in the post treatment biopsies. 1. Veldman-Jones, M. et al. Reproducible, quantitative and flexible molecular sub-typing of clinical DLBCL samples using the NanoString nCounter system. Clin Cancer Res (2014) 2. Flinn I, B.N., Blum KA, et al. A Phase II Trial to Evaluate the Efficacy of Fostamatinib in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma (DLBCL). American Society of Hematology (San Francisco, 2014). 3. Zhang, J. et al. Genetic heterogeneity of diffuse large B-cell lymphoma. Proc Natl Acad Sci U S A 110, 1398-403 (2013). 4. Pasqualucci, L. et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nat Genet 43, 830-7 (2011). Citation Format: Danielle Greenawalt, Kate Byth, Zhongwu Lai, Justin Johnson, Ambar Ahmed, Brian Dougherty, Kenneth Thress, Michael Zinda, Winnie S. Liang, John Carpten, Stephen Fawell, J. Carl Barrett. Whole exome sequencing of pre and post treatment diffuse large B cell lymphoma reveals the mutation spectrum of the relapse/refractory patient population. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1706. doi:10.1158/1538-7445.AM2015-1706