Hakim Bouamar

MicroRNAs miR-125a and miR-125b constitutively activate the NF-κB pathway by targeting the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20)

Constitutive activation of the NF-κB pathway is associated with diffuse large B-cell lymphoma (DLBCL) pathogenesis, but whether microRNA dysfunction can contribute to these events remains unclear. Starting from an integrative screening strategy, we uncovered that the negative NF-κB regulator TNFAIP3 is a direct target of miR-125a and miR-125b, which are commonly gained and/or overexpressed in DLBCL. Ectopic expression of these microRNAs in multiple cell models enhanced K63-linked ubiquitination of proximal signaling complexes and elevated NF-κB activity, leading to aberrant expression of its transcriptional targets and the development of a proproliferative and antiapoptotic phenotype in malignant B cells. Concordantly, genetic inhibition of miR-125a/miR-125b blunted NF-κB signals, whereas rescue assays and genetic modulation of a TNFAIP3-null model defined the essential role of the TNFAIP3 targeting on miR-125a/miR-125b-mediated lymphomagenesis. Importantly, miR-125a/mir-125b effects on TNFAIP3 expression and NF-κB activity were confirmed in a well-characterized cohort of primary DLBCLs. Our data delineate a unique epigenetic model for aberrant activation of the NF-κB pathway in cancer and provide a coherent mechanism for the role of these miRNAs in immune cell activation and hematopoiesis. Further, as miR-125b is a direct NF-κB transcriptional target, our results suggest the presence of a positive self-regulatory loop whereby termination of TNFAIP3 function by miR-125 could strengthen and prolong NF-κB activity.

A capture-sequencing strategy identifies IRF8, EBF1, and APRIL as novel IGH fusion partners in B-cell lymphoma

The characterization of immunoglobulin heavy chain (IGH) translocations provides information on the diagnosis and guides therapeutic decisions in mature B-cell malignancies while enhancing our understanding of normal and malignant B-cell biology. However, existing methodologies for the detection of IGH translocations are labor intensive, often require viable cells, and are biased toward known IGH fusions. To overcome these limitations, we developed a capture sequencing strategy for the identification of IGH rearrangements at nucleotide level resolution and tested its capabilities as a diagnostic and discovery tool in 78 primary diffuse large B-cell lymphomas (DLBCLs). We readily identified IGH-BCL2, IGH-BCL6, IGH-MYC, and IGH-CCND1 fusions and discovered IRF8, EBF1, and TNFSF13 (APRIL) as novel IGH partners in these tumors. IRF8 and TNFSF13 expression was significantly higher in lymphomas with IGH rearrangements targeting these loci. Modeling the deregulation of IRF8 and EBF1 in vitro defined a lymphomagenic profile characterized by up-regulation of AID and/or BCL6, down-regulation of PRMD1, and resistance to apoptosis. Using a capture sequencing strategy, we discovered the B-cell relevant genes IRF8, EBF1, and TNFSF13 as novel targets for IGH deregulation. This methodology is poised to change how IGH translocations are identified in clinical settings while remaining a powerful tool to uncover the pathogenesis of B-cell malignancies.

MicroRNA-21 inhibits p57Kip2 expression in prostate cancer

Backgroundp57Kip2, a cyclin-dependent kinase inhibitor, is considered to be a candidate tumor suppressor gene that has been implicated in Beckwith-Wiedemann syndrome and sporadic cancers. In addition, decreased expression of p57Kip2 protein has been frequently observed in pancreatic, lung, breast, bladder, gastrointestinal tract and prostate cancers. However, p57Kip2 gene mutations are rare in these cancers suggesting that other unknown mechanisms might be at play in reducing its expression. The aim of this study was to investigate the molecular mechanism of down-regulation of p57Kip2 in prostate cancer.FindingsWe observed a significant negative correlation between the expression of p57Kip2 and microRNA-21 (miR-21) in prostate cancer samples and after androgen deprivation with castration in the CWR22 human prostate cancer xenograft model. We report that miR-21 targeted the coding region and decreased p57Kip2 mRNA and protein levels in prostate cancer cells. Conversely, inhibition of endogenous miR-21 by an anti-miR-21 inhibitor strongly induced p57Kip2 expression. Furthermore, we found that knockdown of p57Kip2 reversed the effects of the anti-miR-21 inhibitor on cell migration and anchorage-independent cell growth.ConclusionsOur results indicate that miR-21 is able to downregulate p57Kip2 expression by targeting the coding region of the gene and is also able to attenuate p57Kip2 mediated functional responses. This is the first report demonstrating that p57Kip2 is a novel target of miR-21 in prostate cancer and revealing a novel oncogenic function of this microRNA.

MicroRNA-155 control of p53 activity is context dependent and mediated by Aicda and Socs1

ABSTRACT In biological processes, the balance between positive and negative inputs is critical for an effective physiological response and to prevent disease. A case in point is the germinal center (GC) reaction, wherein high mutational and proliferation rates are accompanied by an obligatory suppression of the DNA repair machinery. Understandably, when the GC reaction goes awry, loss of immune cells or lymphoid cancer ensues. Here, we detail the functional interactions that make microRNA 155 (miR-155) a key part of this process. Upon antigen exposure, miR-155−/− mature B cells displayed significantly higher double-strand DNA break (DSB) accumulation and p53 activation than their miR-155+/+ counterparts. Using B cell-specific knockdown strategies, we confirmed the role of the miR-155 target Aicda (activation-induced cytidine deaminase) in this process and, in combination with a gain-of-function model, unveiled a previously unappreciated role for Socs1 in directly modulating p53 activity and the DNA damage response in B lymphocytes. Thus, miR-155 controls the outcome of the GC reaction by modulating its initiation (Aicda) and termination (Socs1/p53 response), suggesting a mechanism to explain the quantitative defect in germinal center B cells found in mice lacking or overexpressing this miRNA.

D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2

Isocitrate dehydrogenases (IDH) convert isocitrate to alpha-ketoglutarate (α-KG). In cancer, mutant IDH1/2 reduces α-KG to D2-hydroxyglutarate (D2-HG) disrupting α-KG-dependent dioxygenases. However, the physiological relevance of controlling the interconversion of D2-HG into α-KG, mediated by D2-hydroxyglutarate dehydrogenase (D2HGDH), remains obscure. Here we show that wild-type D2HGDH elevates α-KG levels, influencing histone and DNA methylation, and HIF1α hydroxylation. Conversely, the D2HGDH mutants that we find in diffuse large B-cell lymphoma are enzymatically inert. D2-HG is a low-abundance metabolite, but we show that it can meaningfully elevate α-KG levels by positively modulating mitochondrial IDH activity and inducing IDH2 expression. Accordingly, genetic depletion of IDH2 abrogates D2HGDH effects, whereas ectopic IDH2 rescues D2HGDH-deficient cells. Our data link D2HGDH to cancer and describe an additional role for the enzyme: the regulation of IDH2 activity and α-KG-mediated epigenetic remodelling. These data further expose the intricacies of mitochondrial metabolism and inform on the pathogenesis of D2HGDH-deficient diseases.

A Novel TGFβ Trap Blocks Chemotherapeutics-Induced TGFβ1 Signaling and Enhances Their Anticancer Activity in Gynecologic Cancers

Purpose: We investigated the mechanisms of how TGFβ pathway is activated by chemotherapeutics and whether a novel TGFβ trap called RER can block chemotherapeutics-induced TGFβ pathway activation and enhance their antitumor activity in gynecologic cancer. Patients and Methods: An unbiased bioinformatic analysis of differentially expressed genes in 31 ovarian cases due to chemotherapy was used to identify altered master regulators. Phosphorylated Smad2 was determined in 30 paired cervical cancer using IHC. Furthermore, the effects of chemotherapeutics on TGFβ signaling and function, and the effects of RER on chemotherapy-induced TGFβ signaling were determined in gynecologic cancer cells. Results: Chemotherapy-induced transcriptome alteration in ovarian cancer was significantly associated with TGFβ signaling activation. Chemotherapy was found to activate TGFβ signaling as indicated by phosphorylated Smad2 in paired cervical tumor samples (pre- and post-chemotherapy). Similar to TGFβ1, chemotherapeutics were found to stimulate Smad2/3 phosphorylation, cell migration, and markers related to epithelial–mesenchymal transition (EMT) and cancer stem cells (CSC). These TGFβ-like effects were due to the stimulation of TGFβ1 expression and secretion, and could all be abrogated by TGFβ inhibitors including a novel TGFβ trap protein called RER both in vitro and in vivo. Importantly, combination treatment with RER and cisplatin showed a higher tumor inhibitory activity than either agent alone in a xenograft model of ovarian cancer. Conclusions: Chemotherapeutics can stimulate TGFβ1 production and consequently enhance TGFβ signaling, EMT, and CSC features resulting in reduced chemo-sensitivity. Combination therapy with a TGFβ inhibitor should alleviate this unintended side effect of chemotherapeutics and enhance their therapeutic efficacy. Clin Cancer Res; 24(12); 2780–93. ©2018 AACR.

TGF-β signal rewiring sustains epithelial-mesenchymal transition of circulating tumor cells in prostate cancer xenograft hosts.

Activation of TGF-β signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-β signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-β signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII-edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro. Therefore, combined targeting of TGF-β and its backup partner ERK represents an attractive strategy for treating mCRPC patients.

Differential effects of GLI2 and GLI3 in regulating cervical cancer malignancy in vitro and in vivo

Advanced, recurrent, or persistent cervical cancer is often incurable. Therefore, in-depth insights into the molecular mechanisms are needed for the development of novel therapeutic targets and the improvement of current therapeutic strategies. In this study, we investigated the role of GLI2 and GLI3 in the regulation of the malignant properties of cervical cancer. We showed that down-regulation of GLI2, but not GLI3, with an inducible GLI2 shRNA inhibited the growth and migration of cervical cancer cell lines, which could be rescued by ectopic expression of GLI2. GLI2 appeared to support cell growth by regulating the mitosis, but not the apoptosis, of the cervical cancer cells. Mechanistically, these functions of GLI2 were in part mediated by the activation of AKT pathway. Knockdown of GLI2, but not GLI3, also inhibited xenograft growth of cervical cancer cells in vivo. Finally, analysis of TCGA data showed that high levels of GLI2, but not GLI3, conferred a poor prognosis in cervical cancer patients. These observations for the first time suggest that GLI2, but not GLI3, exerts a tumor-promoting role in cervical cancer and may be targeted as a novel therapeutic strategy.GLI transcription factors, which mediate the Hedgehog pathway, are over-expressed in cervical cancer. RNA interference reveals an essential role of GLI2, but not GLI3, in promoting proliferation and migration and xenograft tumor growth of human cervical cancer cells. Mechanistically, the function of GLI2 is mediated by protein kinase B (AKT), and may be targeted as a novel therapeutic strategy.

Isolation, Culture, and Differentiation of Mammary Epithelial Stem/Progenitor Cells from Fresh or Ex Vivo Cultured Human Breast Tissue

In vivo transplantation is the gold standard method for characterization of stem/progenitor cell self‐renewal, tissue regeneration, and tumorigenesis. The method requires an enriched population of stem cells that represent a small fraction of a given tissue. An enriched population of stem/progenitor cells increases the likelihood of engraftment and reduces the number of recipient animals needed for in vivo transplantation. Methods for mammosphere formation by mammary epithelial stem and progenitor cells have been widely adopted for enriching stem/progenitor cells, allowing researchers to study genetic and epigenetic properties, interaction with other cell types, and differentiation and oncogenic transformation. The generation of mammospheres is complex, however, involving many steps and requiring particular skill. Here we describe a detailed mammosphere protocol, including isolation and culture of human primary mammary epithelial stem/progenitor cells and their differentiation and passage in 3D organoid culture. We also describe a protocol for ex vivo culture of fresh human breast tissue for use in assays of clinical treatment. Step‐by‐step instructions detail tissue handling through passage of the stem/progenitor cell‐generated 3D organoids, which can be used to assess the properties, function, and neoplastic transformation of mammary stem/progenitor cells.

Abstract 2424: Hepatocellular carcinoma in the South Texas Latino population: Implications of STEAP2

Introduction: Hepatocellular carcinoma (HCC) is the most common type of liver cancer in adults and the third most common cause of cancer death worldwide; while incidence and mortality rates are two times higher in Latinos, incidence rates are the highest among Latinos in the South Texas region. The genetic and epigenetic events associated with the increased incidence of HCC in this population are largely unknown. We performed whole genome RNA sequencing in paired HCC tumor and adjacent non-tumor tissue total RNA from nine South Texas Latino patients. Analysis of differentially expressed genes revealed significant alterations in pathways associated with oxidative stress; most importantly, we found that the expression of STEAP2 (Six Transmembrane Epithelial Antigen of the Prostate 2) is increased five-fold in HCC tumor tissue compared to adjacent non-tumor tissue. In comparison to a non-Latino population, this finding was unique to South Texas Latinos. STEAP2 is a metalloreductase of iron and copper; reduced iron and copper ions can mediate the production of hydroxyl radicals resulting in increased oxidative stress, which can cause DNA damage and lipid peroxidation. We aim to prove that STEAP2 through regulation of iron and copper homeostasis, and an increase in oxidative stress, will lead to malignant transformation of hepatocytes resulting in tumor progression of HCC, including in obese hosts. Material and Methods: Latino paired HCC and adjacent non-tumor tissues were collected for RNA sequencing, metal ion measurement and oxidative stress markers. STEAP2 RNA and protein expression levels in Latino and Caucasian samples were evaluated by RT-PCR, Western blot, and immunohistochemistry. HCC cell lines (SNU398 and HUH7) with knockdown (KD) and overexpression (OE) of STEAP2 were created to examine the proliferation, migration, anchorage independent growth, and oxidative stress in vitro. Results: Analysis of RNA sequencing data demonstrated the overexpression of STEAP2 in HCC tumors in Latino patients, which were validated by RT-PCR and Western blot data. Lipid peroxidation product, 4-hydroxynonenal, and copper levels were higher in HCC tumor vs. adjacent tissue. KD of STEAP2 in the HCC cell lines decreased proliferation, migration and anchorage independent growth, while OE of STEAP2 increase migration and anchorage independent growth but not proliferation. Conclusions: STEAP2 is specifically overexpressed in HCC tumors in Latinos in comparison to HCC tumors in non-Latino whites and appears to play a malignant-promoting role in HCC cells. Further studies on the role of STEAP2 as a novel tumor promoter in HCC and the mechanisms by which it promotes carcinogenesis are underway. The proposed studies will likely yield mechanistic insights into the molecular mechanisms that drive HCC development and progression in South Texas Latinos and potential therapeutic targets. Citation Format: Carla Zeballos, Hakim Bouamar, Guixi Zheng, Xiang Gu, Yidong Chen, Francisco G. Cigarroa, Lu-Zhe Sun. Hepatocellular carcinoma in the South Texas Latino population: Implications of STEAP2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2424.