Farhad Vesuna

Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.

Oncogenic role of DDX3 in breast cancer biogenesis

Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.

Twist contributes to hormone resistance in breast cancer by downregulating estrogen receptor-α

The role of estrogen receptor-α (ER) in breast cancer development, and as a primary clinical marker for breast cancer prognosis, has been well documented. In this study, we identified the oncogenic protein, TWIST1 (Twist), which is overexpressed in high-grade breast cancers, as a potential negative regulator of ER expression. Functional characterization of ER regulation by Twist was performed using Twist low (MCF-7, T-47D) and Twist high (Hs 578T, MDA-MB-231, MCF-7/Twist) expressing cell lines. All Twist high expressing cell lines exhibited low ER transcript and protein levels. By chromatin immunoprecipitation and promoter assays, we demonstrated that Twist could directly bind to E-boxes in the ER promoter and significantly downregulate ER promoter activity in vitro. Functionally, Twist overexpression caused estrogen-independent proliferation of breast cells, and promoted hormone resistance to the selective estrogen receptor modulator tamoxifen and selective estrogen receptor down-regulator fulvestrant. Importantly, this effect was reversible on downregulating Twist. In addition, orthotopic tumors generated in mice using MCF-7/Twist cells were resistant to tamoxifen. These tumors had high vascular volume and permeability surface area, as determined by magnetic resonance imaging (MRI). Mechanistically, Twist recruited DNA methyltransferase 3B (DNMT3B) to the ER promoter, leading to a significantly higher degree of ER promoter methylation compared with parental cells. Furthermore, we demonstrated by co-immunoprecipitation that Twist interacted with histone deacetylase 1 (HDAC1) at the ER promoter, causing histone deacetylation and chromatin condensation, further reducing ER transcript levels. Functional re-expression of ER was achieved using the demethylating agent, 5-azacytidine, and the HDAC inhibitor, valproic acid. Finally, an inverse relationship was observed between Twist and ER expression in human breast tumors. In summary, the regulation of ER by Twist could be an underlying mechanism for the loss of ER activity observed in breast tumors, and may contribute to the generation of hormone-resistant, ER-negative breast cancer.

Targeting DDX3 with a small molecule inhibitor for lung cancer therapy

Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first‐in‐class small molecule inhibitor, RK‐33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK‐33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3‐overexpressing cells. Importantly, RK‐33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK‐33 impaired Wnt signaling through disruption of the DDX3–β‐catenin axis and inhibited non‐homologous end joining—the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK‐33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

The Twist box domain is required for Twist1-induced prostate cancer metastasis

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial–mesenchymal transition (EMT) that promotes cancer metastasis. Structure–function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Implications: Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential. Mol Cancer Res; 11(11); 1387–400. ©2013 AACR.

Glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) expression correlates with malignant choline phospholipid metabolite profiles in human breast cancer

Altered choline phospholipid metabolism is a hallmark of cancer, leading to malignant choline metabolite profiles consisting of low glycerophosphocholine (GPC) and high phosphocholine (PC) in human breast cancers. Glycerophosphocholine phosphodiesterase (GPC‐PDE) catalyzes the degradation of GPC to free choline and glycerol‐3‐phosphate. The gene(s) encoding for the GPC‐PDE(s) responsible for GPC degradation in breast cancers have not yet been identified. Here, we demonstrate for the first time that the GPC‐PDE encoded by glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) is associated with breast cancer malignancy. Two human breast cancer cell lines (n = 8 and n = 10) and primary human breast tumor samples (n = 19) were studied with combined MRS and quantitative reverse transcription‐polymerase chain reaction to investigate several isoforms of GDPD expression with respect to choline phospholipid metabolite levels. Of the five GDPDs tested, GDPD5 was found to be significantly overexpressed in highly malignant estrogen receptor negative (ER–) compared with weakly malignant estrogen receptor positive (ER+) human breast cancer cells (p = 0.027) and breast tumors from patients (p = 0.015). GDPD5 showed significantly positive correlations with PC (p < 0.001), total choline (tCho) (p = 0.007) and PC/GPC (p < 0.001) levels in human breast tumors. GDPD5 showed a trend towards a negative correlation with GPC levels (p = 0.130). Human breast cancers with malignant choline metabolite profiles consisting of low GPC and high PC levels highly co‐expressed GDPD5, choline kinase alpha (CHKA) and phosphatidylcholine‐specific phospholipase D1 (PLD1), whereas cancers containing high GPC and relatively low PC levels displayed low co‐expression of GDPD5, CHKA and PLD1. GDPD5, CHKA and PLD1 were significantly overexpressed in highly malignant ER– tumors in our patient cohort. Our study identified GDPD5 as a GPC‐PDE that probably participates in the regulation of choline phospholipid metabolism in breast cancer, which possibly occurs in cooperation with CHKA and PLD1. Copyright

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer. We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear β-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer. RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 μM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1. Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.

Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells.

DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.

NZ51, a ring-expanded nucleoside analog, inhibits motility and viability of breast cancer cells by targeting the RNA helicase DDX3.

DDX3X (DDX3), a human RNA helicase, is over expressed in multiple breast cancer cell lines and its expression levels are directly correlated to cellular aggressiveness. NZ51, a ring-expanded nucleoside analogue (REN) has been reported to inhibit the ATP dependent helicase activity of DDX3. Molecular modeling of NZ51 binding to DDX3 indicated that the 5:7-fused imidazodiazepine ring of NZ51 was incorporated into the ATP binding pocket of DDX3. In this study, we investigated the anticancer properties of NZ51 in MCF-7 and MDA-MB-231 breast cancer cell lines. NZ51 treatment decreased cellular motility and cell viability of MCF-7 and MDA-MB-231 cells with IC50 values in the low micromolar range. Biological knockdown of DDX3 in MCF-7 and MDA-MB-231 cells resulted in decreased proliferation rates and reduced clonogenicity. In addition, NZ51 was effective in killing breast cancer cells under hypoxic conditions with the same potency as observed during normoxia. Mechanistic studies indicated that NZ51 did not cause DDX3 degradation, but greatly diminished its functionality. Moreover, in vivo experiments demonstrated that DDX3 knockdown by shRNA resulted in reduced tumor volume and metastasis without altering tumor vascular volume or permeability-surface area. In initial in vivo experiments, NZ51 treatment did not significantly reduce tumor volume. Further studies are needed to optimize drug formulation, dose and delivery. Continuing work will determine the in vitro-in vivo correlation of NZ51 activity and its utility in a clinical setting.

Contributing Factors of Temozolomide Resistance in MCF-7 Tumor Xenograft Models

Vasculature mediated drug resistance in tumors was studied in female SCID mice bearing wild type MCF-7 and adriamycin resistant MCF-7/ADR xenograft using temozolomide (TMZ). A strong tumor growth inhibitory effect of TMZ treatment was 5 observed in MCF-7 tumors during the initial treatment phase with subsequent relapse, but not in MCF-7/ADR tumors. Non-invasive MRI measurements of tumor vascular volume and vascular permeability-surface area product (PS) demonstrated significant reduction of PS in long-term treated MCF-7, but not in MCF-7/ADR tumors. O6-Methylguanine-DNA methyltransferase (MGMT) mRNA, and VEGF expression 10 was analyzed using real-time RT-PCR and ELISA, respectively. No significant changes in MGMT mRNA and VEGF expression were observed in either MCF-7 or MCF-7/ADR tumors. However, in vitro incubation of MCF-7 cells with TMZ did induce the expression of MGMT mRNA. In addition, p53 and p21 levels were scored by immunoblotting. Exposure of cells to TMZ did not affect either the p21 or 15 the p53 expression in both MCF-7 and MCF-7/ADR cells. The absence of these molecular responses to TMZ treatment in MCF-7 tumors in vivo supports the possibility that the onset of cancer drug resistance is associated with reduced PS, which can decrease the delivery of the drug to the cancer cells.