Bakhtiyor Yakubov

Small Molecule Inhibitors Target the Tissue Transglutaminase and Fibronectin Interaction

Tissue transglutaminase (TG2) mediates protein crosslinking through generation of ε−(γ-glutamyl) lysine isopeptide bonds and promotes cell adhesion through interaction with fibronectin (FN) and integrins. Cell adhesion to the peritoneal matrix regulated by TG2 facilitates ovarian cancer dissemination. Therefore, disruption of the TG2-FN complex by small molecules may inhibit cell adhesion and metastasis. A novel high throughput screening (HTS) assay based on AlphaLISA™ technology was developed to measure the formation of a complex between His-TG2 and the biotinylated FN fragment that binds TG2 and to discover small molecules that inhibit this protein-protein interaction. Several hits were identified from 10,000 compounds screened. The top candidates selected based on >70% inhibition of the TG2/FN complex formation were confirmed by using ELISA and bioassays measuring cell adhesion, migration, invasion, and proliferation. In conclusion, the AlphaLISA bead format assay measuring the TG2-FN interaction is robust and suitable for HTS of small molecules. One compound identified from the screen (TG53) potently inhibited ovarian cancer cell adhesion to FN, cell migration, and invasion and could be further developed as a potential inhibitor for ovarian cancer dissemination.

Tissue Transglutaminase Mediated Tumor–Stroma Interaction Promotes Pancreatic Cancer Progression

Purpose: Aggressive pancreatic cancer is commonly associated with a dense desmoplastic stroma, which forms a protective niche for cancer cells. The objective of the study was to determine the functions of tissue transglutaminase (TG2), a Ca2+-dependent enzyme that cross-links proteins through transamidation and is abundantly expressed by pancreatic cancer cells in the pancreatic stroma. Experimental Design: Orthotopic pancreatic xenografts and coculture systems tested the mechanisms by which the enzyme modulates tumor–stroma interactions. Results: We show that TG2 secreted by cancer cells effectively molds the stroma by cross-linking collagen, which, in turn, activates fibroblasts and stimulates their proliferation. The stiff fibrotic stromal reaction conveys mechanical cues to cancer cells, leading to activation of the YAP/TAZ transcription factors, promoting cell proliferation and tumor growth. Stable knockdown of TG2 in pancreatic cancer cells leads to decreased size of pancreatic xenografts. Conclusions: Taken together, our results demonstrate that TG2 secreted in the tumor microenvironment orchestrates the cross-talk between cancer cells and stroma fundamentally affecting tumor growth. Our study supports TG2 inhibition in the pancreatic stroma as a novel strategy to block pancreatic cancer progression. Clin Cancer Res; 21(19); 4482–93. ©2015 AACR.

17β-Estradiol mediates superior adaptation of right ventricular function to acute strenuous exercise in female rats with severe pulmonary hypertension.

17β-Estradiol (E2) exerts protective effects on right ventricular (RV) function in pulmonary arterial hypertension (PAH). Since acute exercise-induced increases in afterload may lead to RV dysfunction in PAH, we sought to determine whether E2 allows for superior RV adaptation after an acute exercise challenge. We studied echocardiographic, hemodynamic, structural, and biochemical markers of RV function in male and female rats with sugen/hypoxia (SuHx)-induced pulmonary hypertension, as well as in ovariectomized (OVX) SuHx females, with or without concomitant E2 repletion (75 μg·kg(-1)·day(-1)) immediately after 45 min of treadmill running at 75% of individually determined maximal aerobic capacity (75% aerobic capacity reserve). Compared with males, intact female rats exhibited higher stroke volume and cardiac indexes, a strong trend for better RV compliance, and less pronounced increases in indexed total pulmonary resistance. OVX abrogated favorable RV adaptations, whereas E2 repletion after OVX markedly improved RV function. E2s effects on pulmonary vascular remodeling were complex and less robust than its RV effects. Postexercise hemodynamics in females with endogenous or exogenous E2 were similar to hemodynamics in nonexercised controls, whereas OVX rats exhibited more severely altered postexercise hemodynamics. E2 mediated inhibitory effects on RV fibrosis and attenuated increases in RV collagen I/III ratio. Proapoptotic signaling, endothelial nitric oxide synthase phosphorylation, and autophagic flux markers were affected by E2 depletion and/or repletion. Markers of impaired autophagic flux correlated with endpoints of RV structure and function. Endogenous and exogenous E2 exerts protective effects on RV function measured immediately after an acute exercise challenge. Harnessing E2s mechanisms may lead to novel RV-directed therapies.

Hypoxia Upregulates Estrogen Receptor β in Pulmonary Artery Endothelial Cells in a HIF-1α–Dependent Manner

&NA; 17&bgr;‐Estradiol (E2) attenuates hypoxia‐induced pulmonary hypertension (HPH) through estrogen receptor (ER)‐dependent effects, including inhibition of hypoxia‐induced endothelial cell proliferation; however, the mechanisms responsible for this remain unknown. We hypothesized that the protective effects of E2 in HPH are mediated through hypoxia‐inducible factor 1&agr; (HIF‐1&agr;)‐dependent increases in ER&bgr; expression. Sprague‐Dawley rats and ER&agr; or ER&bgr; knockout mice were exposed to hypobaric hypoxia for 2‐3 weeks. The effects of hypoxia were also studied in primary rat or human pulmonary artery endothelial cells (PAECs). Hypoxia increased expression of ER&bgr;, but not ER&agr;, in lungs from HPH rats as well as in rat and human PAECs. ER&bgr; mRNA time dependently increased in PAECs exposed to hypoxia. Normoxic HIF‐1&agr;/HIF‐2&agr; stabilization increased PAEC ER&bgr;, whereas HIF‐1&agr; knockdown decreased ER&bgr; abundance in hypoxic PAECs. In turn, ER&bgr; knockdown in hypoxic PAECs increased HIF‐2&agr; expression, suggesting a hypoxia‐sensitive feedback mechanism. ER&bgr; knockdown in hypoxic PAECs also decreased expression of the HIF inhibitor prolyl hydroxylase 2 (PHD2), whereas ER&bgr; activation increased PHD2 and decreased both HIF‐1&agr; and HIF‐2&agr;, suggesting that ER&bgr; regulates the PHD2/HIF‐1&agr;/HIF‐2&agr; axis during hypoxia. Whereas hypoxic wild‐type or ER&agr; knockout mice treated with E2 demonstrated less pulmonary vascular remodeling and decreased HIF‐1&agr; after hypoxia compared with untreated hypoxic mice, ER&bgr; knockout mice exhibited increased HIF‐2&agr; and an attenuated response to E2 during hypoxia. Taken together, our results demonstrate a novel and potentially therapeutically targetable mechanism whereby hypoxia, via HIF‐1&agr;, increases ER&bgr; expression and the E2‐ER&bgr; axis targets PHD2, HIF‐1&agr;, and HIF‐2&agr; to attenuate HPH development.

Abstract 1541: Heterogeneous response to platinum in metastatic ovarian cancer detectable by biodynamic imaging

The mechanisms by which tumors develop multi-drug resistance are not well understood. Biodynamic imaging (BDI) is a label-free assay that detects phenotypic response of ex vivo tissue to chemotherapeutic drugs. In the current study, we used BDI to measure the response of primary and metastatic ovarian tumors to cisplatin. Xenografts from two human ovarian cancer cell lines, A2780 and SKOV3, were grown orthotopically in the ovaries of nude mice and allowed to metastasize in the peritoneal space. Primary and metastatic tumors were harvested and tested for response to 25 μM cisplatin by using BDI. Sensitivity was assessed through selected response biomarkers. Metastatic tumors of the platinum-sensitive A2780 cell line showed 20% decrease in sensitivity to cisplatin compared with the response of the primary tumors, while the less sensitive SKOV3 tumors exhibited a 6% decrease in response of metastatic vs. primary tumors. In parallel, RNA sequencing profiled primary ovarian and metastatic implants. Differential gene expression was detected in primary tumors vs. metastatic sites. Genes upregulated in metastases include TIMP3, WNT2, OSR1, elastin (SKOV3 tumor model) and MMP1, MMP3, MMP10, MET, EGFR, PDGF-C (A2780 tumor model). The study demonstrates the ability of BDI to detect differences in drug sensitivity between primary and metastatic tumors and provides support for the association of metastasis and chemoresistance in ovarian cancer. Supported by NIH R01 EB016582. Citation Format: Daniel Merrill, Hao Sun, Daniela Matei, Bakhtiyor Yakubov, John Turek, David Nolte. Heterogeneous response to platinum in metastatic ovarian cancer detectable by biodynamic imaging. [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 1541.

Abstract 1551: Soluble tissue transglutaminase modulates cellular adhesion to matrix and peritoneal metastasis in ovarian cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Tissue transglutaminase (TG2) is a secreted protein that binds fibronectin (FN) and exerts protein transamidating activity in the presence of Ca2+. We previously reported that TG2 is upregulated in ovarian cancer (OC) cells and enhances intraperitoneal (ip) metastasis. TG2 is secreted abundantly in OC ascites, yet the function of soluble (s) TG2 remains unknown. We hypothesized that sTG2 acts as a permissive factor in the ECM by promoting cancer cell proliferation and adhesion to the matrix. Materials and Methods: Recombinant his6-tagged full length human TG2 and its inactive enzymatic mutant C277S were purified by immobilized metal (Co2+) affinity and anion exchange chromatography. OV90 and A2780 OC cells that do not express endogenous TG2 were used. Solid phase adhesion assay measured adhesion to ECM proteins, MTT assay measured cell proliferation and an ip xenograft model quantified tumor formation and metastasis. Results: S-TG2 enhanced cell adhesion to FN (p = 0.01) and vitronectin (VN, p = 0.01), but not to collagen and laminin. A neutralizing antibody to α5β1 and to αVβ3 blocked sTG2 induced cell adhesion to FN or to VN, respectively, suggesting that sTG2 modulates cell interaction with the matrix via integrins. Digestion of FN with matrix metallo-proteinase 2 blocked OC cell adhesion to digested fragments of FN, while addition of sTG2 restored OC cells’ binding to this protease-digested matrix (p = 0.02). This suggests that the soluble enzyme may facilitate the interaction of OC cells with a protease-remodeled matrix remodeled, similar to the tumor ECM. Enzymatically inactive soluble C277S TG2 enhanced OC cell adhesion to FN to the same extent as wild type TG2. In contrast, an antibody raised against the FN-binding domain of TG2 blocked sTG2 induced OC cell adhesion to FN (p =0.01). These data suggest that the interaction between TG2 and FN, but not the transamidating activity, is important to sTG2-mediated cell interaction with the ECM. When added to conditioned media, sTG2 (2.5ug/mL) increased OC cell proliferation by 20% (p = 0.01). Biweekly ip inoculation of 2.5ug sTG2 increased peritoneal dissemination of OV90 cells in nude mice (11.38 vs. 27.75, p = 0.02), but did not affect tumor volume (412.3 vs. 578.2 mm3, p = 0.34). Conclusions: These data demonstrate that sTG2 increases OC cell adhesion to the ECM and peritoneal metastasis. The key mechanism involved in this process is the interaction between sTG2, FN, and integrins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1551. doi:10.1158/1538-7445.AM2011-1551