Amber Smith

Sorafenib Sensitizes (−)-Gossypol-Induced Growth Suppression in Androgen-Independent Prostate Cancer Cells via Mcl-1 Inhibition and Bak Activation

The natural BH3-mimetic (−)-gossypol shows promising efficacy in ongoing phase II/III clinical trials for human prostate cancer. Here, we show for the first time, that treatment with (−)-gossypol and multikinase inhibitor sorafenib synergistically suppresses the growth of androgen-independent prostate cancer cells (AI-PC) in vitro and in vivo. Our data suggest that sorafenib attenuates (−)-gossypol-induced Mcl-1 upregulation in AI-PCs. In this way, it serves as a potent chemosensitizer to affect cell death. Interestingly, (−)-gossypol and sorafenib induce cell death via two distinct pathways among different AI-PCs; DU145 cells via apoptosis and PC-3 via autophagy. The appointed death pathway may depend on the level of proapoptotic protein Bak, although the level of antiapoptotic protein Bcl-2 plays some role in it. DU145 cells with high Bak level prefer apoptosis induction, whereas PC-3 cells with low Bak prefer the induction of autophagy. Furthermore, inhibiting nondominant death pathways, that is, autophagy in DU145 and apoptosis in PC-3, enhances cell killing by (−)-gossypol/sorafenib combination therapy. Ultimately, our data expose a new action for sorafenib as an enhancer of (−)-gossypol-induced cell growth suppression and reveal a novel cell death mode by Bak activation manners in AI-PCs. These new insights may facilitate the rational design of clinical trials by selecting patients most likely to benefit from the Bcl-2–targeted molecular therapy. Mol Cancer Ther; 11(2); 416–26. ©2011 AACR.

Natural product (−)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1

Musashi‐1 (MSI1) is an RNA‐binding protein that acts as a translation activator or repressor of target mRNAs. The best‐characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin‐dependent kinase inhibitor P21WAF−1. We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA‐binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (−)‐gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti‐tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (−)‐gossypol with the RNA binding pocket of MSI1. We further showed that (−)‐gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (−)‐gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (−)‐gossypol as a potential small molecule inhibitor of MSI1‐RNA interaction, and suggests that inhibition of MSI1s RNA binding activity may be an effective anti‐cancer strategy.

MicroRNA-383 acts as a tumor suppressor in colorectal cancer by modulating CREPT/RPRD1B expression

CREPT (Cell‐cycle‐related and expression‐elevated protein in tumor)/RPRD1B, a novel protein that enhances the transcription of Cyclin D1 to promote cell proliferation during tumorigenesis, was demonstrated highly expressed in most of tumors. However, it remains unclear how CREPT is regulated in colorectal cancers. In this study, we report that miR‐383 negatively regulates CREPT expression. We observed that CREPT was up‐regulated but the expression of miR‐383 was down regulated in both colon cancer cell lines and colon tumor tissues. Intriguingly, we found that enforced expression of miR‐383 inhibited the expression of CREPT at both the mRNA and protein level. Using a luciferase reporter, we showed that miR‐383 targeted the 3′‐UTR of CREPT mRNA directly. Consistently we observed that over expression of miR‐383 shortened the half‐life of CREPT mRNA in varieties of colorectal cancer cells. Furthermore, restoration of miR‐383 inhibited cell growth and colony formation of colon cancer cells accompanied by inhibition of expression of CREPT and related downstream genes. Finally, we demonstrated that stable over expression of miR‐383 in colon cancer cells decreased the growth of the tumors. Our results revealed that the abundant expression of CREPT in colorectal cancers is attributed to the decreased level of miR‐383. This study shed a new light on the potential therapeutic therapy strategy for colorectal cancers using introduced miRNA.

Abstract 2449: Targeting an “undruggable” RNA-binding protein: Discovery of small molecule inhibitors of HuR for novel breast cancer therapy

Post-transcriptional gene regulation is essential for normal development, but when dysregulated, has many implications in disease conditions, including cancer. The RNA-binding proteins (RBPs) are critical trans factors that associate with specific cis elements present in mRNAs, thereby regulating the fate of target mRNAs. Due to the lack of well-defined binding pocket, RBPs have been considered “undruggable targets”. The RBP Hu antigen R (HuR) is overexpressed in many types of cancer, including breast cancer. Elevated cytoplasmic HuR level correlates with high-grade malignancy and serves as a prognostic factor of poor clinical outcome in breast cancer. HuR promotes tumorigenesis by interacting with cancer-associated mRNAs, those mRNAs encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. HuR also modulates the sensitivity of breast cancer cells to chemotherapy. Our hypothesis is that small molecule compounds that disrupt the HuR-mRNA interaction will block HuR function, leading to the decay and reduced translation of mRNAs of the target genes critical for breast cancer cell growth and progression. High throughput screening (HTS) was carried out in several chemical libraries (∼23,000 compounds) using fluorescence polarization (FP) assay and identified a series of initial hits with sub-micromolar inhibitory constants (Ki). Those potential disruptors were then validated by ALPHA assay (Amplified Luminescent Proximity Homogeneous Assay), confirmed by Surface Plasmon Resonance (SPR). In cell-based assays, top hit ST-3 and its optimized analogs, specifically shortened HuR target mRNAs’ half-life and decreased the level of the encoded proteins (Bcl-2, XIAP and Msi1/2). Moreover, those compounds selectively inhibited breast cancer cell proliferation but not normal cells. Knocking down HuR in breast cancer cells attenuated the activity of those HuR-mRNA disruptors. More cell-based assays are carrying out to delineate the mechanism of action. We are also testing the antitumor efficacy of those HuR inhibitors in mouse xenograft models. In conclusion, we identified potential small molecule disrupters of HuR-mRNA interaction for potential novel cancer therapy that inhibited breast cancer with HuR overexpression. Citation Format: Xiaoqing Wu, Lan Lan, Amber Smith, Rebecca Marquez, David Wilson, Steven Rogers, Philip Gao, Scott Lovell, John Karanicolas, Dan Dixon, Jeffrey Aube, Liang Xu. Targeting an “undruggable” RNA-binding protein: Discovery of small molecule inhibitors of HuR for novel breast cancer therapy. [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 2449. doi:10.1158/1538-7445.AM2015-2449

Natural product derivative Gossypolone inhibits Musashi family of RNA-binding proteins

BackgroundThe Musashi (MSI) family of RNA-binding proteins is best known for the role in post-transcriptional regulation of target mRNAs. Elevated MSI1 levels in a variety of human cancer are associated with up-regulation of Notch/Wnt signaling. MSI1 binds to and negatively regulates translation of Numb and APC (adenomatous polyposis coli), negative regulators of Notch and Wnt signaling respectively.MethodsPreviously, we have shown that the natural product (−)-gossypol as the first known small molecule inhibitor of MSI1 that down-regulates Notch/Wnt signaling and inhibits tumor xenograft growth in vivo. Using a fluorescence polarization (FP) competition assay, we identified gossypolone (Gn) with a > 20-fold increase in Ki value compared to (−)-gossypol. We validated Gn binding to MSI1 using surface plasmon resonance, nuclear magnetic resonance, and cellular thermal shift assay, and tested the effects of Gn on colon cancer cells and colon cancer DLD-1 xenografts in nude mice.ResultsIn colon cancer cells, Gn reduced Notch/Wnt signaling and induced apoptosis. Compared to (−)-gossypol, the same concentration of Gn is less active in all the cell assays tested. To increase Gn bioavailability, we used PEGylated liposomes in our in vivo studies. Gn-lip via tail vein injection inhibited the growth of human colon cancer DLD-1 xenografts in nude mice, as compared to the untreated control (P < 0.01, n = 10).ConclusionOur data suggest that PEGylation improved the bioavailability of Gn as well as achieved tumor-targeted delivery and controlled release of Gn, which enhanced its overall biocompatibility and drug efficacy in vivo. This provides proof of concept for the development of Gn-lip as a molecular therapy for colon cancer with MSI1/MSI2 overexpression.

Abstract 1133: Discovery of novel small molecule inhibitors of RNA-binding protein Musashi-1

1) Background and Objective: Musashi-1(Msi1) is an RNA-binding protein that negatively regulates translation by binding to the 3′-UTRs of target mRNAs including Numb, APC and p21WAF-1, key regulators of Notch, Wnt signaling and cell cycle progression, respectively. We aim to identify small molecule inhibitors of Msi1’s mRNA-binding activity thus blocking the growth of a broad range of cancer cells. 2) Methods: Fluorescence polarization assay was used to screen for small molecules that disrupt the binding of Msi1 to its consensus RNA binding site. Hits were validated by surface plasmon resonance, amplified luminescent proximity homogeneous assay and nuclear magnetic resonance. 3) Results: Among ~2,000 small molecules we screened, we found several clusters of compounds that disrupt the Msi1-numb RNA binding potently, including natural products, herbal extracts and fungal metabolites. One of the clusters is the derivatives of the secondary metabolites from Aspergillus nidulans. The top hit, Aza-9, from this cluster was validated by amplified luminescent proximity homogeneous assay. Further validation by surface plasmon resonance and nuclear magnetic resonance indicate that Aza-9 binds to Msi1 directly, in the RNA binding pocket. 4) Discussion and Conclusions: Our study supports the hypothesis that Msi1 inhibition is a viable and effective anti-cancer strategy. More target validation assays will be carried out for the compounds from other clusters. Citation Format: Lan Lan, Amber Smith, Xiaoqing Wu, Anuradha Roy, Ragul Gowthaman, John Karanicolas, Amber D. Somoza, Clay C.C. Wang, Berl Oakley, Roberto De Guzman, Kristi Neufeld, Liang Xu. Discovery of novel small molecule inhibitors of RNA-binding protein Musashi-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1133. doi:10.1158/1538-7445.AM2017-1133

Abstract 1460: Tumor suppressor miR-137 inhibits colorectal cancer progression by negatively regulating cancer stem cell marker, Musashi-1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Colorectal cancer is the 3rd most common cause of cancer-related deaths for men and women in the United States. Understanding the mechanisms involved in colon cancer initiation and progression requires immediate attention. Multiple regulatory processes are required to maintain normal cell homeostasis, including microRNA (miRNA) regulation. miRNAs are predicted to target 60% of all mRNAs, therefore, providing substantial regulatory power of many cellular processes. The goal of this project is to identify and characterize dysregulated miRNAs in colorectal cancer. We previously demonstrated that the colon cancer stem cell marker, Musashi1 (Msi1), is negatively regulated by miR-137. Msi1 is an RNA binding protein over-expressed in a variety of cancers, including colon cancer. Msi1 promotes tumor growth by positively regulating both Notch and Wnt signaling. Notch and Wnt signaling play important roles in colorectal cancer initiation and progression. We hypothesize that miR-137 acts as a tumor suppressive miRNA by negatively regulating Msi1 and subsequent Notch and Wnt signaling. Using Taqman miRNA assay, we discovered that miR-137 expression is decreased in approximately 84% of rectal tumor samples as compared to paired normal rectal mucosal samples (n=68). Consistent with the clinical data, our colon cancer cell lines have decreased expression of miR-137 as compared to a normal colon epithelial cell line. Restoration of miR-137 reduced colon cancer proliferation, measured by colony formation and tumorsphere assays. Furthermore, miR-137 restoration decreased Msi1 which resulted in subsequent decreased Notch and Wnt signaling, measured by quantitative Real-Time PCR and Western blotting. In order to understand the effect of miR-137 on tumor growth, HCT-116 colon cancer cells were stably transduced with a doxycycline-inducible miR-137 lentiviral expression vector. In vivo studies were carried out whereby athymic nude mice were subcutaneously injected with either negative control miRNA or miR-137 stable cell lines. After tumors reached approximately 50 mm3 in size, mice were fed doxycycline to induce expression of miR-137 or negative control miRNA. Induction of miR-137 expression significantly decreased tumor growth in our xenograft model by approximately 50% (n=10). In conclusion, our research demonstrates miR-137 as a potent tumor suppressive miRNA that is lost in colorectal cancer and negatively regulates the colon cancer stem cell marker, Musashi-1. Citation Format: Amber R. Smith, Rebecca Marquez, Bryan Tsao, Lan Lan, Surajit Pathak, Xiao-Feng Sun, Kristi Neufeld, Liang Xu. Tumor suppressor miR-137 inhibits colorectal cancer progression by negatively regulating cancer stem cell marker, Musashi-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1460. doi:10.1158/1538-7445.AM2014-1460

Abstract 5240: Epigenetic dysregulation of miR-196b in breast cancer

Dysregulation of microRNA (miRNA) expression levels is a major cause of cancer development. miRNA expression profiles can determine tumor type, patient prognosis, reoccurrence and treatment outcome, with greater accuracy as compared to mRNA expression profiles. The expression of miRNAs can be epigenetically dysregulated by aberrant DNA methylation and histone deacetylation in breast cancer cells. Furthermore, one third of miRNA promoters are targets for aberrant methylation in breast cancer. Identifying miRNAs that are epigenetically silenced in breast cancer will provide new insight into breast cancer development as well as define novel signatures for predicting therapeutic response to epigenetic modifying agents. New studies have shown that using transient low doses of demethylating agents, such as decitabine (DAC), result in sustained changes in major cancer cell signaling pathways, therefore reducing tumorigenicity and cancer stem cell self-renewal. The goal of this project is to identify epigenetically-silenced miRNAs that can be reactivated upon low dose treatment of DAC. miR-196b has been shown to inhibit metastasis by targeting HOXC8. Using online datasets, we determined miR-196b is hypermethylated in breast cancer patient samples compared to normal breast samples. We used Taqman miRNA assays to measure the expression of miR-196b in breast cancer patient samples and found it was down-regulated in tumor samples compared to paired normal breast tissue. miR-196b is also down-regulated in MDA-MB-231 cells. We treated MDA-MB-231 cells with low dose (100 nM) DAC for 3 days and found miR-196b was upregulated 5-fold compared to DMSO control. The methylated CpG content of miR-196b-associated CpG island was measured using bisulfite sequencing DNA purified from DMSO and DAC treated cells. The miR-196b CpG loci in the DAC treatment was demethylated by 33% compared to DMSO control. Therefore, reactivation of miR-196b is associated with demethylation of miR-196b CpG loci. Interestingly, fourteen days after ending DAC treatment, miR-196b re-expression continued to increase up to 13-fold above DMSO control. In addition, we found that exogenously re-introduced mature miR-196b into MDA-MB-231 breast cancer cells resulted in upregulation of endogenous primary miR-196b. This suggests a novel positive feedback loop that allows miR-196b to regulate its own expression, possibly through epigenetic mechanisms. Future studies will involve identifying the mechanism associated with this re-expression, determining whether miR-196b regulates additional gene promoters, and the affect this has on breast cancer metastatic phenotype. Citation Format: Rebecca T. Marquez, Amber R. Smith, Bryan Tsao, Liang Xu. Epigenetic dysregulation of miR-196b in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5240. doi:10.1158/1538-7445.AM2014-5240