Given that most cancer patients die from metastatic disease, there is an urgent need to develop drugs that inhibit the spread of tumors. Studies show that the motuporamines activate the small GTPase RhoA to prevent cancer cell migration, but little is known about the mechanism of action of dihydromotuporamine C (Motu33) and its synthetic derivative Motu-(CH2)-33. In the present study, I investigated the biomolecular processes of these compounds in Drosophila by reducing the gene dose of positive and negative regulators of actin dynamics. Consistent with previous findings, reduced gene dose of Rho1 (the Drosophila RhoA ortholog) attenuates motuporamine activity confirming that RhoA/Rho1 are targeted by these compounds. Actin-myosin contraction is controlled by the Rho1-ROCK-myosin regulatory light chain (MRLC) pathway. Reduced gene dose of the myosin binding subunit of myosin phosphatase, which is a negative regulator of the Rho1-ROCK-MRLC pathway, encourages motuporamine activity indicating that the motuporamines stimulate actin-myosin contraction. Rho1 also activates diaphanous (dia) to control actin polymerization. Surprisingly, reduced gene dose of dia facilitates motuporamine activity suggesting that the motuporamines act on dia in a Rho1-independent manner. Reduction in gene dose of the Drosophila Rac orthologs Rac1 and Mtl enhances motuporamine activity. In contrast, motuporamine activity is unaffected by reduced gene dose of slingshot (ssh) which acts to trigger actin severing and depolymerization. Since ssh is directly regulated by Rac1, the enhanced activity of motuporamines under reduced Rac1 and Mtl gene dose may reflect an indirect mode of action on the Rac GTPases leading to increased Rho1 activity. In all, these findings indicate that motuporamines act through Rho1 and diaphanous to regulate actin-myosin contractility and actin polymerization and may be a promising novel therapy to deter cancer cell migration.

The capability for expansive tumor growth is described as a key hallmark of cancer. Discovering the mechanisms driving this proliferative capacity could advance new targeted therapies to inhibit tumor growth, recurrence, and metastasis. With the aim of identifying new components of the regulatory circuit driving tumor growth, we studied the role of Chaperonin-Containing TCP1 (CCT or TRiC) in promoting breast cancer. CCT is a cytosolic protein folding macromolecular complex composed of eight subunits (CCT1-8) that interacts and assists in the folding of a number of substrates including oncoproteins and mutated tumor suppressors. Recent findings from our lab and others revealed that the second subunit, CCT2, is specifically upregulated and causes uncontrolled growth in breast cancer as shown by CCT2 overexpression and depletion studies. In 3D and 2D cultures of luminal A breast cancer cells, T47D and MCF-7, CCT2 overexpression promoted the formation of tumor-like spheroids, increased cell proliferation, and cell cycle progression, and promoted anchorage-independent growth. Importantly, CCT2 gene expression correlated with increased levels of cell cycle promoting genes like MYC, CCND1 (cyclin D1), and CDKN2 (CDK2) and potentially could serve as a hub for converging pro-tumor cell signaling pathways. These findings led to the premise that the upregulation of CCT2 expression in cancer cells could be triggered by stress conditions such as uncontrolled growth in nutrient-poor in vivo conditions. In support, depletion of CCT2 by 40-50% did not affect luminal A breast cancer cells grown in unstressed, nutrient-rich cell culture conditions but did inhibit in vivo tumor growth of metastatic breast cancer cells in mice. Hence, we investigated whether increased CCT2 could be linked to other stress conditions such as those that result in the development of drug resistance to cancer therapies. Inhibitors of the cell cycle mediators, CDK4/6, such as Palbociclib, show significant patient benefit that is offset by the eventual development of resistance. To determine whether CCT2 could have a role in promoting resistance to CDK4/6 inhibition, we treated control and CCT2-overexpressing T47D and MCF-7 cells with Palbociclib. We noted that CCT2-overexpressing cells displayed increased resistance to Palbociclib as compared to control cells and upon long-term culture with Palbociclib recovered faster, with two-four fold increases in cell number after 30 days. These cells retained increased proliferative capacity after treatment cessation, and, in response to Palbociclib treatment, increased CCT2 levels beyond that observed in basal cultures. This increase in CCT2 gene expression correlated with increases in CCND1 and CDKN2, which suggests a possible compensatory mechanism that enables resistance to CDK4/6 inhibition. These findings do not rule other cell cycle independent compensatory pathways such as increased metabolic activity that bears further study. Our findings suggest that CCT2 may be selectively increased in cancer cells in response to stress conditions and, thereby, has an important role in supporting uncontrolled growth in cancer progression and promoting drug resistance. CCT2 could thus serve as a prognostic and therapeutic target for multi- targeted inhibition that would overcome drug resistance and enhance patient treatment outcomes

Pancreatic cancer overall has a poor five-year survival rate of less than 8%. Well-tolerated regimens and immune cell infiltration to promote anti-cancer treatments are major challenges. Studies described herein leveraged testing different combinations/doses of polyamine targeting inhibitors to further understand the impact of targeting key polyamine pathway mediators in pancreatic ductal adenocarcinoma (PDAC). Placebo controls were compared to groups treated with difluoromethylornithine (DFMO, a polyamine biosynthesis inhibitor of ornithine decarboxylase), different polyamine transport inhibitors, and compound combinations in strategies for polyamine blockage therapy (PBT). Informatic analyses showed that the dysregulation of key polyamine pathway mediators are associated with poor patient prognosis. A PBT strategy combining DFMO and Trimer44NMe was well tolerated over extended treatment in a syngeneic, immunocompetent mouse model. This in vivo PBT strategy significantly reduced tumor size and increased survival in comparison to DFMO alone. Results also showed increased infiltration of CD86+ immune cells into the PDAC tumor in DFMO and/or the PBT combination, although further investigation is needed to understand their impact on the PDAC microenvironment. A second PBT strategy that combined DFMO and a non-polyamine-based inhibitor (GW5704) was effective against PDAC cells in vitro and in immunodeficient mice, but not in the immunocompetent mouse tumor model used above. Only tumors treated with DFMO exhibited downregulation of MYC and significantly increased infiltration of T cells. Overall effectiveness of a PBT strategy was dependent on the drug that was combined with DFMO. MYC suppression was linked to improved survival and immunomodulatory changes in the pancreatic tumor microenvironment. Overall, the present study points to DFMO being an immunomodulatory agent, and a need for further understanding of DFMO-based polyamine-inhibitory therapeutic strategies in PDAC. Further studies are need to understand how PBT treatment can improve in vivo therapeutic outcomes in part through immune modulation in the tumor microenvironment.

Epigenetic silencing is often altered in cancer and is a target for drug discovery. Unbiased screens in live cells are performed to identify potential novel targets of epigenetic therapy, and these screens have identified drugs that were not previously recognized to be involved in epigenetic reactivation of gene silencing such as cardiac glycosides and a CDK9 inhibitor. Recently, our lab performed a whole genome siRNA screen in combination with DNMT inhibition. One of the top targets revealed in this screen was the splicing factor SF3B1. SF3B1 is a well-known crucial splicing factor and is mutated in several cancers. However, its role in epigenetic regulation has not been well studied. I propose SF3B1 is a novel target for epigenetic therapy in cancer. In the YB5 colon cancer cell line where GFP is under the control of a methylated CMV promoter, I validated the screen results and found 0%, 1.0% and 5.3% GFP+ cells after treatment with siControl, siSF3B1 or the DNA methyltransferase inhibitor decitabine (DAC), respectively. DAC and siSF3B1 were synergistic, inducing 17.2% GFP+ cells. This synergy was also seen in an additional live cell assay and with other SF3B and SF3A family proteins. RNA-Seq analyses showed 423 genes upregulated by siSF3B1, 430 genes induced by DAC, and 1190 induced by the combination. siSF3B1 resulted in aberrant splicing of 695 genes, but there were only 27 genes overlapping between splicing alterations and gene expression changes, suggesting different mechanisms. Genes regulated upon siSF3B1 treatment were enriched for the TATA motif in their promoters, and the TATA-Box binding protein (TBP) was among the genes differentially spliced after siSF3B1. DNA methylation analyses showed demethylation synergy between siSF3B1 and DAC. Finally, the effects of siSF3B1 were phenocopied by treatment with the pan-SF3B inhibitor Pladienolide B (PB). GFP was reactivated in two separate colon cancer cell lines upon treatment with PB with synergistic activation when combined with DAC in YB5 cells. Thousands of genes were regulated and alternatively spliced with PB treatment alone, and among the differentially spliced genes was TBP. Furthermore, PB treatment with DAC induced demethylation significantly more than with DAC treatment alone. Genes regulated upon SF3B1 loss and inhibition were enriched for p53 target genes. Indeed, there was reduced cell proliferation and cell cycle arrest when SF3B1 was inhibited. This study demonstrates that the splicing factor SF3B1 has unexpected effects on gene transcription and targeting SF3B1 is synergistic with DNA methylation inhibition suggesting clinical potential for the combination. / Biomedical Sciences

Epigenetic deregulation contributes significantly to the development of multiple human diseases, including cancer. While great effort has been made to elucidate the underlying mechanism, our knowledge on epigenetic regulation is still fragmentary, an important gap being how the diverse epigenetic events coordinate to control gene transcription. In the first part of our study, we demonstrated an important link between Snail-mediated transcriptional control and epigenetic regulation during cancer development. Specifically, we found that the highly conserved SNAG domain of Snail sequentially and structurally mimics the N-terminal tail of histone H3, thereby functions as a molecular “hook”, or pseudo substrate, for recruiting histone lysine specific demethylase 1 (LSD1) repressor complex to the E-cadherin promoter. Furthermore, we showed that Snail and LSD1 are both required for E-cadherin repression and EMT induction, and their expression is highly correlated with each other in multiple human tumor tissues. Our findings have important clinical ramifications in that compounds mimicking the SNAG domain may disrupt Snail-LSD1 interaction and inhibit EMT and metastasis. In the second part of our study, we designed a batch of compounds based on the structure of the SNAG domain and are currently screening for candidates capable of competing with SNAG peptide for LSD1 binding. In addition, we applied a peptide pulldown/mass spectrometry-coupled analysis to identify SNAG-interacting proteins, among which are many chromatin enzymes and modulators. Functional characterization of these proteins will help to elucidate the Snail-mediated epigenetic regulation process. In the third part of our study, we found that Snail interacts with poly(ADP-ribose) polymerase 1 (PARP1) through a potential pADPr-binding motif and is subject to poly(ADP-ribosyl)ation, which can stabilize the Snail-LSD1 complex for enhanced PTEN suppression under DNA damage condition. Our findings added another layer to the delicate Snail transcriptional machinery, and indicated that PARP inhibitors may be applied in combination with conventional chemotherapies to target cancers with high expression of Snail and LSD1. In summary, we demonstrated that Snail cooperates with multiple epigenetic machineries to induce EMT as well as survival of tumor cells. Our findings contribute to a better appreciation of Snail-mediated epigenetic network as well as diversification of therapeutic strategies against cancer.

Identification of therapeutically relevant molecules is necessary for the advancement of non-viral reprogramming of human cells for regenerative medicine. We have developed a novel non-viral model system that transforms primary human dermal fibroblasts into cells with induced regeneration competence (iRC). Low oxygen-mediated effects of fibroblast growth factor FGF2 lead to an increased cellular lifespan with a two fold increase in population doublings before senescence, remaining non-tumorigenic when injected into SCID mice while maintaining regeneration competence. This system allows us to study molecules that participate in increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by hybridization array, RT-PCR, and Western blots revealed upregulation of the arginine methyltransferase PRMT8 in iRC cells, challenging the paradigm that PRMT8 is solely expressed in brain tissue at the plasma membrane. Possibly leading to the erroneous conclusions that PRMT8 is brain specific at the plasma membrane is the fact that PRMT8 has several mRNA variants and protein isoforms. Here, I report expression of a novel PRMT8 variant in human dermal fibroblasts. Essential participation of PRMT8 in cellular proliferation was identified as a novel function for this enzyme through siRNA-mediated knockdown in both non-tumorigenic and tumorigenic cell lines. While other members of the PRMT family have known roles in cell cycle progression, I show for the first time that PRMT8 expression is reduced in both natural senescence and by premature induction of replicative senescence using sub-cytotoxic levels of hydrogen peroxide, implicating a correlation between PRMT8 expression and cell cycle progression. However, PRMT8 overexpression causes no significant change in the number of population doublings or the amount of time spent in culture prior to senescence, and does not alter the expression of key cell cycle regulatory genes. These results suggest that maintenance of PRMT8 expression is critical for cellular proliferation, but overexpression of PRMT8 alone is not sufficient to increase cellular lifespan. I determined that oxygen is the primary mediator of PRMT8 upregulation in the iRC system and therefore investigate histone occupancy of the PRMT8 promoter at hypoxia response elements. Through this analysis, I found bivalent occupancy regardless of culture conditions, indicating that PRMT8 maintains a state of poised readiness for transcriptional accessibility. The mechanism by which PRMT8 participates in cellular proliferation was investigated through binding partner identification. A binding partner of endogenous PRMT8 is identified here for the first time as FGF2 using co-IP and mass spectrometry. As iRC cells demonstrate a unique phenotype that uncouples the mechanisms of increased lifespan from tumorigenesis, I investigated the feasibility of PRMT8 as a cancer biomarker by mining publically available data in light of our own. I showed that PRMT8 is not only expressed in a variety of cancers, but that its expression is amplified. Moreover, PRMT8 expression significantly correlates to patient survival in specific cancers, strengthening the feasibility of this molecule as a biomarker. Aberrant expression of most PRMT family members has been described in various cancers, and specific PRMT variants are currently being used as prognostic markers. As such, I analyzed variant-specific PRMT8 expression in primary cancer cell lines and show that tumorigenic glioblastomas express PRMT8 mRNA variant 2. These data suggest that PRMT8 is a viable candidate for further study as a prognostic cancer biomarker, specifically for brain cancer.

Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation. Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling. Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL-C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases. Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells.

Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation. Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling. Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL-C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases. Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells.

Whole genome doubling (WGD) occurs early in tumorigenesis and generates genetically unstable tetraploid cells that fuel tumor development. Cells that undergo WGD (WGD+) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can subsequently be exploited therapeutically, is unclear. Using sequencing data from ~10,000 primary human cancer samples and essentiality data from ~600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent on spindle assembly checkpoint signaling, DNA replication factors, and proteasome function than WGD– cells. We also identify KIF18A, which encodes for a mitotic kinesin, as being specifically required for the viability of WGD+ cells. While loss of KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD– cells, its loss induces dramatic mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results reveal new strategies to specifically target WGD+ cancer cells while sparing the normal, non-transformed WGD– cells that comprise human tissue.

In cells undergoing stress, the p53 transcription factor is stabilized and activates the expression of numerous genes contributing to p53-mediated tumor suppression. One p53 target gene is Mdm2, which encodes an oncoprotein that binds and ubiquitinates p53 for proteasomal degradation, thus limiting the amplitude and duration of the p53-mediated stress response. Our lab recently discovered that Mdm2 phosphorylation by ATM and c-Abl regulates the DNA damage response and tumorigenesis in mice. AKT has also been found in transfection studies to phosphorylate Mdm2 at serine residues 166 and 186 (mouse S163 and S183) to alter p53 activity. However, the physiological significance of Mdm2 phosphorylation by Akt remains unknown. Therefore, I generated Mdm2S163A or Mdm2S183A mice expressing mutant Mdm2 incapable of being phosphorylated by Akt. In contrast with our previous studies, Akt phosphorylation of Mdm2 does not alter spontaneous tumorigenesis or the DNA damage response to ionizing radiation. However, Akt phosphorylation of Mdm2-S183 (but not -S163) upregulates nuclear localization of Mdm2, destabilizes p53, and reduces p53-mediated senescence in response to elevated levels of reactive oxygen species (ROS). To examine the effects of Mdm2-S183 phosphorylation on p53 tumor suppression, I utilized three different mouse models of ROS-induced cancer. Increased levels of p53 and senescence in Mdm2S183A mice yielded reduced tumorigenesis in an activated Ras model of lung cancer, a phorbal ester-induced skin cancer model, and a diethylnitrosamine-induced model of hepatocellular carcinoma. Since AKT is also important regulator of cell metabolism, I explored the impact of the Mdm2-S183 allele on metabolic functions. Mdm2 phosphorylation by Akt reduced glucose metabolism via glycolysis in vitro, and reduced insulin tolerance in mice, without altering glucose tolerance and glucose-stimulated insulin secretion. Collectively, these findings document a unique physiologic role for the AKT-Mdm2-p53 signaling axis in regulating cell growth and tumorigenesis.