<p> <i>Chromobacterium violaceum</i> (CV) produces a violet color pigment known as Violacein. It has been reported that violacein has anticancer activity. This compound is produced by CV a gram-negative facultatively anaerobic bacterium found in soil and water environmental samples. The purpose of this study was to determine the effect of purified violacein on select cancer cell lines. Violacein used in this study was purified from CV strain (14N23), a strain isolated from environmental samples collected in the Tennessee Copper Basin. The previous reports used a crude extract preparation of violacein; thus, it was of interest to determine the effect of the pure compound on cancer cell growth was similar to that of the crude extracts. The compound purified following the method of Mehta, et al. was exposed to cancer cells and cell death assessed using the Alamar Blue procedure. It was found that violacein had no effect on A549, BT549, and PC3 cancer cell growth; however, there was a significant effect on Colo-320 cancer cells. It was concluded that further studies are required to assess the effect of violacein on enzymes and proteins involved in the cancer cell apoptotic pathways. Such studies will explain why cancer cell death was observed in certain cancer cells and not others.</p>

<p> Women with Type 2 diabetes (T2D) have a 49% increase in breast cancer related mortality compared to women without T2D. Epidemiological studies report that increased endogenous insulin levels and increased insulin receptor (IR) expression are associated with poor survival in breast cancer patients. Therefore, it is essential to investigate the role of endogenous hyperinsulinemia on breast cancer progression. Presented in this thesis are contributions to understanding the effect of insulin in a mouse model of hyperinsulinemia (MKR mouse). First, data is shown that highlights the significant increase in primary MVT-1 tumors and pulmonary metastasis in the MKR mouse compared to Wild Type mice. The studies presented show that the primary tumors from the MKR mice have significantly higher Vimentin protein expression compared to primary tumors from control mice. Next, the studies determine that silencing Vimentin expression in the tumor cells leads to either decreased number of pulmonary metastasis in the hyperinsulinemic mice. The work in this thesis also establishes a novel immunodeficient hyperinsulinemic (Rag/MKR) mouse model that enabled the study of the effects of endogenous insulin on the progression of human cancer cells. The hyperinsulinemia of the Rag/MKR mice promoted a significant increase in tumor growth of MDA-MB-231 and LCC6 cells. The knockdown of the insulin receptor in the LCC6 cells led to primary tumors that were significantly smaller in both the hyperinsulinemic Rag/MKR and Rag/WT control mice compared to the tumors from the LCC6 control cells. Finally, it is shown for the first time that the knockdown of the IR promotes a reversal of the epithelial-mesenchymal phenotype by repressing mesenchymal markers and re-expressing epithelial markers in the LCC6 insulin receptor knockdown tumors. The data presented in this thesis highlight a potential contribution to the understanding of the role of insulin in the setting of hyperinsulinemia and provide potential targets for therapy to improve survival in women with breast cancer and hyperinsulinemia.</p>

<p> Although castration-resistant prostate cancers no longer respond to anti-androgen therapies, the androgen receptor (AR) is still required to promote tumor survival. However, the signaling pathways downstream of AR that promote this survival are not well known. We recently identified an AR-dependent survival pathway whereby AR induction of integrin &alpha;6&beta;1 and adhesion to laminin activates NF-&kappa;B/RelA signaling and Bcl-xL. This pathway acts in parallel with the PI3K/Akt pathway in Pten-null tumor cells such that combined inhibition of both PI3K and integrin &alpha;6&beta;1 is required to effectively kill tumor cells adherent to laminin. However, PTEN-null castration-resistant tumors were not effectively killed by this combination. I discovered that BNIP3, a hypoxia-induced BH3-only, pro-mitophagic Bcl-2 family member, is induced by androgen in castration-resistant cells through integrin &alpha;6&beta;1 and HIF1&alpha;. Furthermore, castration-resistant cells adherent to laminin were much more efficient at inducing autophagy in response to androgen. Androgen blocked the ability of the PI3K inhibitor PX866 to kill castration-resistant tumors, but this was reversed by loss of BNIP3. Although BNIP3 was dispensable for androgen-induced autophagy, its mitophagy function was required for BNIP3 to promote resistance to PX866. Thus, enhanced hypoxia signaling in cooperation with AR/&alpha;6&beta;1/HIF1&alpha; signaling on laminin in castration-resistant cells drives the expression of BNIP3 and enhances autophagy, both of which contribute to PX866 resistance through induction of mitophagy.</p><p>

<p> The majority of breast cancers express the estrogen receptor (ER+) and are treated with anti-estrogen therapies, particularly the inhibitor tamoxifen. However, many women treated with tamoxifen develop resistance, leading to metastatic disease, which is responsible for the majority of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen sensitive and resistant breast cancer cells, a patient derived tamoxifen-resistant xenograft model, and genome-wide transcription and translation studies, we show that tamoxifen resistance is mediated by selective mRNA translational reprogramming. Tamoxifen resistant translation is mediated by increased expression of translation factor eIF4E, increased mTOR activity to promote eIF4E availability, and increased MNK activity to promote eIF4E Ser209 phosphorylation. Tamoxifen re-sensitization is restored only by reducing eIF4E expression or mTOR activity and blocking MNK1-directed eIF4E phosphorylation. Of the translationally upregulated mRNAs specific to tamoxifen resistant cells, we show that Runx2, which encodes a regulator of ER signaling that antagonizes estrogen responses and promotes breast cancer metastasis, significantly increases tamoxifen resistance and restores sensitivity when silenced. Moreover, tamoxifen resistant but not sensitive patient ER+ breast cancer specimens demonstrate strongly increased levels of mTOR and MNK activity and eIF4E protein. eIF4E levels, availability and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through translatome reprogramming.</p><p>

<p> Osteosarcoma (OS) is the most common type of solid bone cancer, mainly arising in children and young adults, and remains the second leading cause of cancer-related death in this age group. Chemotherapy resistance underlying latent recurrence and metastasis represent major contributors to poor outcome for many cancer patients especially those with OS. Tumor hypoxia is an essential element intrinsic to most solid tumor microenvironments and is associated with resistance to therapy and a malignant phenotype, while metastatic dissemination is dependent on a cells ability to resist anoikis, i.e., programmed cell death in the absence of attachment to an extracellular matrix. We sought to better characterize hypoxia and anoikis resistance in human OS using established and novel patient-derived OS cells and OS animal models with the long-term goal of identifying and validating targetable signaling pathways. We show that hypoxia-inducible factors (HIFs), canonical proteins associated with the hypoxic response, are present and can be induced in human OS cells. We demonstrate that the Wnt/&beta;-catenin signaling pathway, a key pathway in OS pathogenesis, is down-regulated in response to hypoxia in OS cells, and that this appears to result from both HIF-dependent and HIF-independent mechanisms. Hypoxia promotes resistance of human OS cells to standard chemotherapy, which is mitigated by treatment with Wnt/&beta;-catenin signaling inhibitors. Using an anchorage-independent growth model, we show that anoikis-resistant OS subpopulations have altered growth rates, increased resistance to standard chemotherapies, and display distinct changes in gene expression and DNA methylation. Finally, we validate the use of two FDA-approved epigenetic therapies predicted by expression profiling in both inhibiting anchorage-independent growth and sensitizing anoikis-resistant OS cells to chemotherapy. In summary, despite the heterogeneity of human OS, our work suggests that unique and effectively targetable signaling pathways underlie the phenotypic consequences in response to hypoxia and anoikis resistance.</p><p>

Breast cancer tops the list of new cancer cases and is predicted to be the second leading cause of cancer deaths in women in 2012. The primary objective of the present study was to provide insights into the molecular mechanisms underlying the aggressive growth and metastasis of triple-negative and basal-like breast cancers. To study increased growth and invasive behavior in triple-negative and basal-like breast cancers we utilize both an interesting and relevant cell culture model and examination of human tissue. S-phase kinase-associated protein 2 (SKP2) plays an important role in cell cycle regulation by targeting p27 for degradation. The cyclin-dependent kinase (CDK) inhibitor p27 regulates G1/S transition by binding cyclin/CDK complexes and abrogating its activity. By targeting p27 for degradation, SKP2 frees the complexes needed to progress into the S phase of the cell cycle. Evaluation of SKP2 expression in TMX2-28 revealed significantly higher levels than in other breast cancer cell lines. Despite the high levels of SKP2 expression, p27 protein was not reduced. However, levels of the Serine 10 phosphorylated form of p27 (pSer10p27), which has been associated with increased proliferation rates, was found to be increased. Furthermore, suppression of SKP2 completely eliminated the pSer10p27 and slowed cycle progression confirming the role of SKP2 in the aggressive growth of TMX2-28 cells. Assessment of mRNA from 30 frozen human breast cancers demonstrated that SKP2 is more highly expressed in ER&agr;-negative and basal-like breast cancers. Immunohistochemical analysis of 188 breast cancers and 50 benign reduction mammoplasty tissues confirmed that SKP2 is more highly expressed in ER&agr;-negative breast cancers and for the first time demonstrated that triple-negative breast cancers are more likely to overexpress SKP2 than are non-triple-negative, but still ER&agr;-negative, tumors. In contrast to some previous reports, we did not observe an inverse relationship between SKP2 and p27 expression. Only 11% of tumors expressed high SKP2 and low p27, while 32% of tumors had high SKP2 and high p27. Although no significant relationship between SKP2 and p27 expression was observed in human breast cancers, a significant positive relationship was discovered between SKP2 and pSer10p27. Furthermore, high levels of SKP2 and pSer10p27 were observed significantly more often in ER&agr;-negative and triple negative breast tumors than in ER&agr;-positive breast cancers. Based on these results and those of the cell culture experiments showing complete elimination of pSer10p27 after suppression of SKP2 it appears that levels of pSer10p27 may be a better indicator of SKP2-dependent p27 degradation than are levels of p27. Therefore, that inhibiting SKP2 in triple-negative breast cancers expressing high levels of both SKP2 and pSer10p27 regardless of p27 levels may be a valid therapeutic approach. We determined that TMX2-28 lack MMP-1 mRNA, and MMP-2/MMP-9 protein expression; each of which is important in protease-dependent invasion. Furthermore, TMX2-28 cells have low expression of other genes key to protease-dependent invasion including Slug, Zeb 1, Zeb 2, Vimentin, Fibronectin and N-cadherin. RhoA is a member of the Rho superfamily of GTPases that acts as a molecular switch to control signal transduction and is critical to the amoeboid invasion mechanism. TMX2-28 cells have high expression of protease-independent invasion genes such as RhoA, ROCK 1, ROCK 2, and E-cadherin. Finally, treating TMX2-28 cells with a RhoA pathway inhibitor or an shRNA targeting RhoA significantly reduces their invasiveness. These data suggest that TMX2-28 cells use a RhoA-dependent, proteolytic-independent invasion mechanism. Collectively, the data presented here demonstrate the roles of SKP2 and RhoA in triple-negative and basal-like breast cancers, making both genes, as well as their pathways, desirable therapeutic targets. (Abstract shortened by UMI.)

Glioblastoma Multiforme (GBM) is the most common brain cancer and one of the most fatal forms of cancer overall. The average survival time is 10-14 months, and less than 10% of patients survive more than 5 years after diagnosis. It is characterized by extreme vasculature, chemo/radioresistance, and invasiveness into the normal brain. The current standard of care, which includes surgical removal of tumor, radiation, and the chemotherapeutic agent temozolomide, initially stunt tumor growth. Nevertheless, the tumor invariably rebounds and the patient succumbs to the disease. Therefore, there is an urgent need to develop new therapies for this devastating disease. YKL-40 is one of the most over-expressed proteins by GBM cells, and is elevated in the serum of patients with GBM. YKL-40 is implicated in a host of inflammatory diseases and has been shown to play a major role in the maturation of some cells of the immune system, especially macrophages. Thus, it has been suggested that YKL-40 may act as a prognostic biomarker for cancer and inflammatory disease. However, little is known about the role of YKL-40 in relation to cancer development and progression, and more work needs to be done to validate it as a biomarker or as a therapeutic target. It was the goal of the work described herein to uncover some of the key molecular mechanisms of GBM development and progression in the hopes of offering new therapeutic targets. Using a wide variety of in vitro and in vivo techniques, the role of a secreted glycoprotein YKL-40 in GBM was probed. It was demonstrated that YKL-40 enhanced angiogenesis, radioresistance, and progression of GBM cells. Moreover, inhibition of YKL-40 in mouse models markedly arrested tumor growth and vascularization, lending support to the idea of YKL-40 as a therapeutic target. Finally, YKL-40 drove GBM cells into a mesenchymal phenotype, where the tumor cells act as mural-like cells, supporting tumor vasculature networks. Hopefully, the results from these studies will offer the rationale to develop drugs against YKL-40 and potentially extend the lives of patients with this terrible disease.

The root of the Tibetan plant Rhodiola crenulata is part of eastern traditional medicine. Studies have suggested that members of the Rhodiola genus display anticancer properties. In this study we examine the effect of R. crenulata in a cellular model of invasive breast cancer, this disease being the second cause of cancer death among women in the US. Deregulation of the Wnt/β-catenin pathway has been frequently observed in breast cancers and appears to have a key role in the transformation of benign cells to a malignant form. Although mutations of the Wnt growth factor are rarely observed in cancer, the Wnt signaling pathway is often up-regulated by either mutations that result in stabilization of β-catenin or by hypermethylation and subsequent loss of expression of Wnt signaling antagonists like secreted Frizzled-Related Protein 1 (SFRP1) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008) (Hanahan and Weinberg 2000; Miyoshi, Rosner et al. 2002; Reya and Clevers 2005; Suzuki, Toyota et al. 2008). We used an engineered cell line in which SFRP1 expression has been knocked down. These cells were derived from 76NTert cell line, an immortalized human mammary epithelium cell line. The resulting 76NTert-siSFRP1 cells display a mesenchymal-like phenotype, invasive behavior and are more resistant to apoptosis triggered by anchorage independent conditions, or anoikis. Additionally we used a highly invasive estrogen receptor negative (ER-), progesterone receptor negative (PR-), Her2/neu negative (triple negative) breast cancer cells line MDA-MB-231. Treatment of MDA-MB-231 and 76NTert-siSFRP1 cells with an extract of R. crenulata inhibited migration and invasion of both cell types, as compared to untreated cells. Furthermore, R. crenulata sensitizes cells to anoikis but does not increase γ-irradiation induced cell death. We provide evidence that death induced by R. crenulata does not occur through the inhibition of an epithelial-to-mesenchymal transition (EMT). Taken together, our initial results suggest R. crenulata as a potential therapeutic agent for breast cancer patients with mutations in the Wnt/β-Catenin signaling pathway. Additionally, we present evidence that R. crenulata exerts its anti-metastasis effect by inhibiting cell migration and increasing cell attachment to a substrate. We demonstrate that this effect occurs by R. crenulata's modulation of the Rho GTPase effector ROCK1. We further show evidence that R. crenulata's effect on ROCK is not limited to cancer cells (in vitro), but also affects isolated and intact embryonic tissues. We discovered that treatment of embryos with R. crenulata can cause a spina bifida phenotype, suggesting (1) that compounds in R. crenulata may prove detrimental to a developing embryo, and (2) the active compounds in R. crenulata may prove useful in the study of developmental anomalies that lead to conditions such as spina bifida. More importantly, our results suggest that pregnant women should avoid taking R. crenulata-containing supplements during pregnancy. Compounds in R. crenulata may be contraindicative to the pregnancy and cause injury to a developing fetus. The information provided may help health providers offer better advice on natural supplements to expecting mothers. We also characterized and identified multiple R. crenulata compounds and report predicted protein targets for these compounds. Finally, we provide evidence that R. crenulata affects cancer cell metabolism and suggest potential protein targets of the chemical components of this extract. This study provides new information that will help dissect the mechanisms of action of the R. crenulata compounds and possible synergies amongst them.