Metastasis is one of the main causes of death in cancer patients. Metastatic melanoma is a death sentence, as chemotherapeutic agents have a 5% success rate or do not extend survival beyond 10 months. The lack of effective chemotherapeutic agents for treating metastatic melanoma indicates a dire need to identify new drug targets and develop new therapies. Our lab has previously shown that the kinase activity of Abelson family of non-receptor tyrosine kinases (c-Abl and Arg) is elevated in invasive breast cancer cell lines as compared to non-invasive cell lines. Previous studies from our lab have shown that Abl kinases are convergent point of ErbB2 and Src Kinases in melanoma cells and Abl kinases promote invasion by an undefined mechanism. Although Abl kinases promote invasion, it is not known whether they are important for metastastic potential. For the first time, we report that Abl kinases promote melanoma cell proliferation, survival, matrigel-invasion and single-cell 3D invasion. To investigate the mechanism by which Abl kinases promote invasion, we found out that active c-Abl transcriptionally upregulates MMP-1, and using rescue approaches we show that c-Abl promotes invasion via a STAT3àMMP-1 pathway. In contrast, active Arg drives invasion in a STAT3-independent manner, and upregulates the expression of MMP-3 and MT1-MMP, in addition to MMP-1. We also found that Abl kinases promote invasion via lysosomal degradation of a metastasis suppressor, NM23-H1 by activating lysosomal cathepsins B and L, which directly cleave and degrade NM23-H1. Furthermore, c-Abl and Arg are activated in primary melanomas and cAbl/Arg activity is inversely correlated with NM23-H1 expression both in primary melanoma and human melanoma cells. We also demonstrate, for the first time that active Abl kinases promote metastasis in vivo, as inhibition of c-Abl/Arg with nilotinib, dramatically inhibits lung colonization/metastasis in a mouse model using two different melanoma cell lines. In summary, we identify Abl kinases as critical, novel, drug targets in metastatic melanoma, and our data indicate that nilotinib may be useful in preventing metastasis in a select group of patients, harboring active Abl kinases.

Pain from bone metastases is multifaceted with clinical descriptors including ongoing pain, hypersensitivity to external stimuli and intermittent episodes of breakthrough pain characterized as a sudden and abrupt onset of severe pain on a background of well-controlled pain. Moreover, cancer-induced bone pain remains inadequately managed due to a myriad of side effects associated with the current pain relieving regimens, which primarily rely on administration of opiates. Despite advances made in cancer therapeutics, these patients experience an inferior quality of life with incapacitating pain with limited daily activities. Development of long-term novel, non-opiate mechanism-based therapeutics with limited side effects is considered beneficial in elevating the patients' quality of life. First part of this dissertation encompasses the role of p38 MAPK in a mouse model of cancer-induced bone pain in which breast cancer cells were injected and sealed into the femur. Our data demonstrated that both acute and prolonged inhibition of p38 MAPK blocked cancer-induced spontaneous pain but had no effect on the evoked pain indicating important differences in mechanisms mediating ongoing pain as opposed to evoked pain. Undermanaged control of breakthrough pain is attributed to poor understanding of underlying mechanisms and how they may differ from ongoing pain due, in part, to lack of a pre-clinical model in which these mechanisms can be studied. We have established a rat model of cancer-induced bone pain to examine ongoing pain and pain relief using conditioned place preference paradigm as well as breakthrough pain using palpation-induced conditioned place aversion. We have shown that while peripheral afferent input from the tumor-bearing tibia mediates cancer-induced ongoing pain and initiation of breakthrough pain, it does not contribute to the maintenance of breakthrough pain. These data suggest that molecular targets mediating these two mechanisms may be different. This hypothesis was confirmed by our findings in this model that acute blockade of interleukin-6 blocked movement-evoked breakthrough pain in tumor-bearing rats, but failed to block tumor-induced ongoing pain. Hence, we provide a platform to manipulate treatments that can be given alone or in combination with opiates in such a way that patients receive adequate control of breakthrough pain.

Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin inhibitors. I hypothesize that the anti-tumor effect of mTOR inhibitors in mantle cell lymphoma is mediated by an increase in manganese superoxide dismutase (MnSOD) protein expression and accumulation of hydrogen peroxide (H₂O₂). Findings indicate that the rapamycin-induced cytostatic effect is characterized by increased levels of MnSOD and H₂O₂, and is necessary for the full growth inhibitory effect of rapamycin. Furthermore, over-expression of MnSOD elevated the level of H₂O₂ and increased sensitivity to MnSOD. Treatment with rapamycin resulted in a loss of serine 473 phosphorylation of AKT and increased levels of MnSOD were found to be due to inhibition of the mTORC2 complex. These results are the first to suggest that long term treatment of MCL cells with rapamycin inhibits the mTORC2 complex. By understanding the key signaling molecules and affected pathways in the anti-tumor effects of mTOR inhibitors, we may be able to identify additional predictive markers to improve the therapeutic value, or study drug combinations that will enhance the effect of ROSinduced cytotoxicity. A retrospective study utilizing samples from lymphoma patients receiving standard anthracycline-based therapies, identified single nucleotide polymorphisms in oxidative stressrelated genes associated with survival. Individuals carrying minor allele SNPs in myeloperoxidase (MPO) and an aldo-keto reductase (AKR1C3) were found to be associated with shorter time to disease progression and death. This data suggest that some patients may benefit from a different therapy than the current standard of care and that regulation of the redox environment plays a role in aggressive lymphoma treatment response.

Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 60,000 annual diagnoses in the U.S. and approximately 500,000 worldwide. About 90% of these individuals receive radiation therapy, and salivary hypofunction and xerostomia occur in 60-85% of these patients due to irreversible damage to the salivary glands. Current preventative and palliative care fail to improve quality of life, accentuating the need for regenerative therapies. Stem/progenitor-cell based therapies have been proposed to regenerate the irradiated glands; however, the identity of stem and progenitor cells in the adult salivary glands has remained somewhat elusive. Moreover, it is unclear how salivary progenitors respond to radiation and whether they can be stimulated to effectively reinstate salivary function. The second chapter of the present study describes the development of a label-retaining assay in salivary glands using EdU. The label-retaining cells (LRCs) identified in murine salivary glands have proliferative potential in vitro and expressed markers of putative salivary progenitors, such as Keratin 5, Keratin 14, and c-Kit. Interestingly, LRCs were still present 30 days following radiation, when chronic loss of saliva is evident. The significance of these findings lies in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining salivary gland homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. In the following chapter, we show that a unique population of murine salivary gland LRCs undergo compensatory proliferation in response to radiation. The initiation of compensatory proliferation is tightly associated with inactivation of the kinase aPKCζ and increased nuclear localization of YAP. This part of the study provides novel insights into the regulation of function of salivary gland progenitors, which can be utilized for the development of therapeutic agents to treat salivary hypofunction. Finally, the last chapter describes how the mechanisms found to initiate compensatory proliferation in acinar LRCs as a response to radiation are involved in the regeneration of salivary glands with IGF-1. Administration of IGF-1 post-radiation restores salivary function in mice, but the mechanisms of regeneration are still unknown. Here, we show that IGF-1 requires aPKCζ to restore saliva production. Further, IGF-1 inhibits nuclear translocation of Yap in an aPKCζ-dependent fashion. We propose that a tightly regulated balance in the levels of aPKCζ and Yap in acinar LRCs has to be maintained in order to restore function following radiation. In conclusion, the findings from this study provide new knowledge in regards to the regulation of function of salivary progenitors during a state of injury (by radiation) and during regeneration (with IGF), and offer potential targets of study for the development of new therapeutics for salivary gland dysfunction. Future studies will determine whether aPKCζ and Yap can be effectively targeted in salivary progenitors to restore salivary function in head and neck cancer patients who receive radiation therapy.

In the presented study, we identified that SRSF3 controls the alternative splicing of CPEB2 and consequently promotes a metastatic phenotype in triple negative breast cancer (TNBC). TNBC causes thousands of deaths annually, frequently due to a lack of effective treatments and a high rate of metastasis in patients. Alternative splicing has been found to be dysregulated in numerous cancers, while splicing factors such as SRSF3 are variably expressed. In this study we performed a siRNA panel to screen potential splicing factors, then used specific siRNA to study the effect of its knockdown on cellular function. These results showed that SRSF3 encourages the production of the pro-metastatic isoform of CPEB2, which contributes the aggressive phenotype of the tumor. We utilized numerous methods to measure the metastatic function of cultured TNBC cells to determine if SRSF3 strongly promoted the metastatic function. These data showed that siRNA reduction of SRSF3 was able to reduce the metastatic potential of cancer cells. These findings suggest that SRSF3 has great potential as a therapeutic measure to reduce and minimize the aggressiveness of TNBC tumors.

p53 is mutated in 50% of all human cancers, and up to 70% of lung cancer. Mutant p53 is usually expressed at elevated levels in cancer cells and has been correlated with a poor prognosis. Cancer cells that express mutant p53 show an increase in oncogenic phenotypes including an increase in growth rate, resistance to chemotherapeutic drugs, and an increase in motility and tumorigenicity to name a few. We have identified several genes involved in cell growth and survival that are upregulated by expression of common p53 mutants: NFκB2, Axl, and epidermal growth factor receptor (EGFR). The aim of this study was to determine the role NFκB2, Axl, and EGFR play in mutant p53’s gain of function (GOF) phenotype and to determine a mechanism for upregulation of mutant p53 target gene upregulation. Inhibition of mutant p53 in various cancer cell lines using RNAi in the form of transient siRNA transfection or stable shRNA cell line generation caused a decrease in the gain of function ability of those cells in the form of reduced chemoresistance, reduced cell growth and motility, and a reduction in tumor formation. Additionally, inhibition of NFκB2, Axl, and EGFR also showed similar effects. Promoter deletion analysis of the NFκB2 promoter did not show a specific mutant p53 response element needed for mutant p53 mediated transactivation. Similarly, deletion of the p53/p63 binding site on the Axl promoter did not inhibit mutant p53 transactivation. Sequence analysis of the NFκB2, Axl, and EGFR promoters revealed several transcription factor binding sites located throughout the promoters. ChIP analysis of mutant p53 and the promoter-specific transcription factor binding revealed that in the presence of mutant p53, individual transcription factor binding is increased to the NFκB2, Axl, and EGFR promoters as well as an increase in acetylated histone binding. This data suggests that mutant p53 promotes an increase in transcription by inducing acetylation of histones via recruitment of transcription factors to the promoters of mutant p53 target genes.

In the United States, more than 130,000 people will be diagnosed with colorectal cancer (CRC) each year and an estimated 50,000 people will die from the disease. Standard of care (SOC) therapies for CRC combine multiple cytotoxic chemotherapeutic drugs. These combinations have varying degrees of effectiveness and can often result in significant patient morbidity. For second recurrence patients, the multi-kinase inhibitor, regorafenib, is an approved agent, but is often poorly tolerated at current doses. In the current study, we propose to develop therapeutic regime of combining agents with modest toxicity profiles: curcumin and sildenafil with regorafenib. Using clinically achievable enterohepatic drug concentrations (~2.0 μM), our laboratory has shown that both sildenafil and curcumin interact to synergistically down regulate the expression of multiple cyto-protective molecular chaperones and kill CRC cells in a greater than additive manner in vitro and suppress the growth of colon cancer tumors in vivo. In this study, the expression of PDGFRb and PDGF in the plasma was increased in colon tumor bearing mice previously exposed to curcumin and sildenafil. Further, the in vitro killing potential of curcumin and sildenafil was shown to be reduced in evolved tumor cells from these mice. The purpose of this study was to determine whether down regulation of PDGFRb using regorafenib would increase the lethality of curcumin and sildenafil in colorectal cancer cell lines in vitro and in vivo. In the current study, we have shown that with the [2μM curcumin + 2μM sildenafil + 2μM regorafenib] drug combination reduces of cyto-protective proteins, enhances cytotoxicity and creates a carnage of CRC cells in a greater than additive in vitro. The combination also suppressed growth of colon cancer tumors in vivo, when compared to curcumin and sildenafil alone. In addition we have shown that the [2μM curcumin + 2μM sildenafil + 2μM regorafenib] drug combination can modulate immune checkpoint proteins in vitro. These results suggest that this drug combination may enhanced the anti-tumor efficacy of anti-PD-1 and anti-CTLA4 antibodies.

Glioblastoma (GBM) is the most common and aggressive type of brain cancer, with an average life expectancy of 15 months. The standard of care for GBM, surgery accompanied by radiation and chemotherapy (temozolomide-TMZ), has not changed in over 10 years illustrating the need for new and efficacious treatments. Therefore, it is imperative to improve our knowledge of GBM physiology to understand the mechanisms driving recurrence and chemoresistance so that more effective therapeutic options can be developed. Mitochondria-cell communication is key to monitor and maintain both mitochondrial and cellular health, and signaling events on the outer mitochondrial membrane (OMM) have emerged as a crucial signal integration site for cellular responses. Consequently, proteins on the OMM are crucial to determining cellular survival and dictating organelle physiology. Thus, the goal of our current study is to evaluate OMM proteins to determine how alterations in organelle regulation may impact CNS tumor biology. We first measured the concentrations of Bcl-2 family proteins on mitochondria from ten continuous GBM cell lines and correlated the protein levels to IC50values of genotoxic agents TMZ and irinotecan. We found that Bcl-2 levels corresponded to chemoresistance, while increased Bim concentrations promoted chemosensitivity. In contrast to our studies in gynecological cancers, the concentrations of the pro-dysfunction OMM scaffold protein Sab had no impact on chemosensitivity of the GBM cell lines, despite diminished Sab expression in GBM patients. However, we identified a novel truncated variant of Sab in the GBM cell lines. We found that GBM cells expressing only full-length Sab had a slower proliferation rate than those with the variant, which could be attributed to increased glycolysis in GBM cells expressing the Sab variant. To determine if the lack of Sab-mediated apoptosis was consistent across CNS tumors, we analyzed publicly-available patient data and found that Sab expression is down-regulated in neuroblastoma patients, a pediatric malignancy responsible for 12% of childhood cancer deaths. We found that that inhibiting Sab-mediated signaling in human neuroblastoma (SH-SY5Y cells) enhanced oxidative phosphorylation in a pyruvate dehydrogenase-dependent manner, increased BCl-2 levels (pro-survival), decreased Bim concentrations (pro-apoptotic), and promoted chemoresistance. Furthermore, examination of additional neuroblastoma cells derived from CNS tumors revealed that Sab levels correspond to proliferation rate, metabolic phenotype, and chemosensitivity. Our studies demonstrate the importance of OMM signaling in CNS tumor physiology and emphasizes the importance of cellular context to the outcomes of OMM signaling events.

Lung cancer is the leading cause of cancer-related death and the second most diagnosed cancer in the United States. Unfortunately, many patients either do not have any common mutations for which there are already targetable agents, or they eventually become resistant to these compounds. As such, there is a high demand for new, effective methods of treating this disease as well as predicting patient prognosis and potential benefit from chemotherapy. In this work, numerous strategies for treating lung cancer are explored. The first method described here is through the use of a pan-early 2 factor (E2F) inhibitor, HLM006474, which is shown to synergize with paclitaxel in non-small cell lung cancer (NSCLC). Next, we explored the creation and utilization of an E2F signature that is prognostic and predictive of early-stage NSCLC patient benefit from adjuvant chemotherapy (ACT). The third project examined possible targets to enhance sensitivity to cisplatin in NSCLC lacking Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma receptor tyrosine kinase (ALK) fusions (triple-negative), for which cisplatin is one of the few treatment options. These studies led to the identification of a kinase that is overexpressed in NSCLC and whose knockdown sensitizes cells to platinum agents.

Most human genes undergo alternative splicing, and many abnormal splicing processes are associated with human diseases. However, the molecular relationship between alternative splicing and tumorigenesis is not well understood. Here, we identified novel Krüppel-like factor 4 (KLF4) splicing variants produced by exon skipping in human cancer cell lines as well as colon tumor tissues. To elucidate the mechanism involved in KLF4 alternative splicing, we developed KLF4 minigene system and found that RNA binding motif protein 5 (RBM5) plays an important role in KLF4 splicing, as assessed by gain and loss of functional studies. Several anti-tumorigenic compounds were also tested for KLF4 splicing. Interestingly, sulindac sulfide restored wild type KLF4 (KLF4L) expression and this is mediated by dephosphorylation of RBM5. Another splicing variant, small KLF4 (KLF4S), localizes in the cytoplasm and nucleus, and antagonizes transcriptional activity of wild type KLF4. Our data suggest that RBM5 plays a pivotal role in the alternative splicing of KLF4, and these splicing variant forms may impact tumorigenesis.