Browsing by Author "Counter, Christopher M"

Telomeres are protein-DNA structures at the ends of eukaryotic chromosomes. The DNA portion is comprised of double-stranded and single-stranded G-rich repetitive DNA. The protein portion is anchored by the "shelterin complex" composed of six proteins. Inappropriate DNA repair and telomere length dysregulation result in cell cycle arrest, genome instability, and carcinogenesis. Thus, this DNA/protein structure protects telomere ends and regulates telomere length.

The shelterin component TRF1, a double-stranded telomeric DNA binding protein, was found to bind accessory protein PinX1 at mitosis. Given this, I investigated the effect of reducing PinX1 level on cell cycle progression and apoptosis. I found that reducing PinX1 expression with shRNA, as assessed by immunoblot, led to delayed entry into mitosis and elevated levels of apoptosis in human cells. These results indicated that PinX1 plays an important role in mitosis progression and cell viability.

Intriguingly, binding of PinX1 to TRF1 at mitosis increased the stability of the latter. Moreover, PinX1 binds to the same site on TRF1 as the protein TIN2, which can suppress degradation of TRF1 by inhibiting poly ADP-ribosylation of TRF1 by the enzyme tankyrase. Collectively, these results suggested that TIN2 might be released from TRF1 to promote the binding of PinX1 on TRF1 at mitosis. Given that proteins are often regulated in the cell cycle by phosphorylation, I investigated whether TIN2 was phosphorylated at mitosis. To this end, I performed phospho-proteomic analysis of human TIN2, which revealed two phosphorylated residues, serines 295 and 330. Both sites were phosphorylated specifically during mitosis, as detected by two independent approaches, namely Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2 in vitro and in vivo. The identification of these specifically timed post-translational events during the cell cycle demonstrates the mitotic regulation of TIN2 by phosphorylation. However, as expressing non-phosphorylatable mutants of TIN2 failed to reveal any overt phenotypes, the consequences of these phosphorylation events remain to be determined.

Lastly, the TRF1-related double-stranded telomeric DNA binding protein, TRF2, was shown to associate with another shelterin component, POT1. POT1 forms heterodimer with TPP1 to bind single-stranded telomeric DNA. Previous research found that mutations of POT1 with reduced binding affinity to either TRF2 or to TPP1 cause distinct phenotypes. To determine whether similar separation-of-function mutants could be generated to dissect the function of POT1s in mice, which are encoded by two genes, Pot1a and Pot1b, I screened a panel of substitution mutants of mPOT1a for loss of binding to mTRF2 and mTPP1. These studies revealed that mPOT1a does not bind mTRF2, but the association with mTPP1 could be disrupted.

In summary, the described studies have shed insight into the complexity of shelterin regulation, and in particular, highlighted protein-protein interactions and post-translational modifications.

Telomeres are the ends of chromosomes which are composed of repetitive DNA sequence and telomere associated proteins. In C. elegans, the protein F39H2.5 was found to associate with the telomere, regulating both telomere length and genomic integrity. F39H2.5 is a member of the β-CASP family of proteins that are known to possess nuclease activity on DNA substrates. I thus sought to address whether any of the human β-CASP family proteins associated with telomeres. Here I show that hSnm1B localized to the telomere indirectly, via interaction with the double-stranded telomere binding protein TRF2. The terminal 37 amino acids of hSnm1B are necessary and sufficient for binding TRF2, and moreover that binding to TRF2 stabilized hSnm1B protein by preventing ubiquitination. In the absence of exogenous TRF2 this domain acted as a degron, promoting protein instability. I thus termed the domain the Protection And INstability (PAIN) domain. I hypothesize that TRF2 binding ensures that hSnm1B will only accumulate at telomeres by preventing the degradation of hSnm1B. However, hSnm1B stability appears to be further regulated, as telomere specific DNA damage stabilized hSnm1B independent of the PAIN domain. Thus, it appears that the telomere associated protein, hSnm1B, is regulated by protein stability in a manner that is both dependent and independent of the PAIN domain.

Telomeres are DNA-protein structures that protect eukaryotic chromosome ends from illegitimate recombination and degradation. Telomeres become shortened with each cell division unless telomerase, a reverse transcriptase, is activated. In addition to playing a protective role of chromosome ends, telomeres and telomere binding proteins are also essential for regulating telomere length and telomerase access. The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage and negatively regulating telomere length. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein-interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded DNA (dsDNA) binding protein TRF2. I sought to determine which of these functions--ssDNA, TPP1, or TRF2 binding--was required for POT1-mediated telomere localization, protection, and length regulation. Using separation-of-function POT1 mutants deficient in one of POT1's three functions, I found that binding to TRF2 fosters robust loading of POT1 onto telomeric chromatin and regulates telomere length, but is dispensable in the protection of telomeres. Although it remains unclear what role TPP1 binding plays in telomere length regulation, I found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1-mediated protection of telomeres, possibly by excluding RPA from telomeres.

Missense oncogenic mutations in the RAS genes are found in around 20% of all human cancers, which are known to be tumorigenic. Despite scores of different oncogenic RAS mutations detected in human cancers, these mutations have distinct patterns, often with a specific set of mutations unique to each cancer type. As RAS mutations can initiate cancer, elucidating the mechanism underlying RAS mutation patterns could inform on the origin of cancer. While capturing the moment cancer begins is not tractable in humans, the process of establishing RAS mutation patterns is recapitulated in mice exposed to the environmental carcinogen urethane, which induces lung tumors driven by one specific Ras mutation. I therefore captured the mutation spectrum of Ras genes after urethane exposure, which revealed that this bias appeared to be a product of the mutagenic preference of the carcinogen as well as high Kras expression. However, when endogenous Kras expression was increased, this mutational bias was shifted to other mutations. I show that in this setting a p53-dependent selection for an optimal signaling level becomes the dominant factor in the bias towards a specific Kras mutation. Collectively, these findings suggest that a multifactorial process shapes the mutational spectrum of RAS genes in cancer.

The protein Snm1B binds to the telomere binding protein TRF2 to help protect telomeres from DNA damage during S phase. In addition, Snm1B protects DNA from agents that induce interstrand crosslinks (ICLs), lethal lesions that covalently attach the opposite strands of DNA together. To elucidate how Snm1B performs these functions I performed a yeast two-hybrid screen to identify proteins binding to the C-terminus of Snm1B. From this screen I identified PSF2 (GINS2), a member of the tetrameric protein complex GINS, to bind Snm1B. The GINS complex is required for replication initiation and elongation. Of interest, the knockdown of PSF2 sensitizes cells to ICL inducing agents. I therefore tested the interaction of PSF2 and Snm1B by co-immunoprecipitation from human cells and discovered that PSF2 binds to two regions of Snm1B. Deletion of the first of these regions inhibited the ability of Snm1B to co-immunoprecipitate with the protein Mus81, a structure specific endonuclease that is required to form double strand breaks (DSBs) as an intermediate in ICL repair. Deletion of the second binding region reduced the ability of PSF2 to localize Snm1B onto chromatin. These data support a role for an interaction of Snm1B with PSF2 in ICL repair.

The small GTPases HRAS, NRAS and KRAS are mutated in approximately one-third of all human cancers, rendering the proteins constitutively active and oncogenic. Lung cancer is the leading cause of cancer deaths worldwide, and more than 20% of human lung cancers harbor mutations in RAS, with 98% of those occurring in the KRAS isoform. While there have been many advances in the understanding of KRAS–driven lung tumorigenesis, it remains a therapeutic challenge. To further this understanding and assess novel approaches for treatment, I have investigated two aspects of Kras–driven tumorigenesis in the lung:

(I) Despite nearly identical protein sequences, the three RAS proto-oncogenes exhibit divergent codon usage. Of the three isoforms, KRAS contains the most rare codons resulting in lower levels of KRAS protein expression relative to HRAS and NRAS. To determine the consequences of rare codon bias during de novo tumorigenesis, we created a knock-in Krasex3op mouse in which synonymous mutations in exon 3 converted codons from rare to common. These mice had reduced tumor burden and fewer oncogenic mutations in the Krasex3op allele following carcinogen exposure. The reduction in tumorigenesis appeared to be a product of rare codons affecting both the oncogenic and non–oncogenic alleles. Converting rare codons to common codons yielded a more potent oncogenic allele that promoted growth arrest and enhanced tumor suppression by the non-oncogenic allele. Thus, rare codons play an integral role in Kras tumorigenesis.

(II) Lung cancer patients exhale higher levels of NO and iNOS-/- mice are resistant to chemically induced lung tumorigenesis. I hypothesize that NO promotes Kras–driven lung adenocarcinoma, and NOS inhibition may decrease Kras–driven lung tumorigenesis. To test this hypothesis, I assessed efficacy of the NOS inhibitor L–NAME in a genetically engineered mouse model of Kras-driven lung adenocarcinoma. Adenoviral Cre recombinase was delivered into the lungs intranasally, resulting in expression of oncogenic KrasG12D and dominant-negative Trp53R172H in lung epithelial cells. L–NAME treatment was provided in the water and continued until survival endpoints. In this model, L–NAME treatment decreased tumor growth and prolonged survival. These data establish a potential clinical role for NOS inhibition in lung cancer treatment.

Genetic variants in Leucine Rich Repeat Kinase 2 (LRRK2) are implicated in multiple diseases, most notably Parkinson’s disease (PD). These variants range in effect from protecting from disease to causing disease. Of the thousands of possible disease-associated genetic variants in the LRRK2 gene, function has only been assigned to a few. Exploiting new functional and bioinformatic approaches, herein, we developed an unbiased pipeline working from the genetic variant back to function, rather than starting with disease status and working back to a candidate variant. We interrogated thousands of missense variants for potential to impact LRRK2 function from both a PD-enriched cohort and broader non-disease related cohort utilizing the combinatorial, computational prediction tool REVEL tailored to predict classifications of rare missense variants. Using newly minted whole-genome sequencing libraries, we identified hundreds of novel LRRK2 variants across a range of ethnicities with high probability of damaging LRRK2-protein function. We performed structural modeling to predict outcome of the altered function of the missense variant on LRRK2 function, and identified top candidates to evaluate biochemically. Additionally, we identified two novel LRRK2 haplotypes with important implications in PD. These findings anchored on two novel assays that were developed to further investigate LRRK2 protein function and intricacies of variant-driven alterations. The first, a high-throughput biomarker-based assay to screen novel LRRK2 variants for functional impact, and the second, a proximity-labeling proteomics assay to uncover network changes associated with functional genetic variation. We developed three novel single-molecule immunoassays (SiMoA) on the Quanterix SR-X platform to evaluate LRRK2 variant functional consequences, particularly centered around the trans-phosphorylation of pT73-Rab10. We also screened our novel variant candidates for another main readout of LRRK2 enzymatic function, autophosphorylation of pS1292-LRRK2. We identified novel variants in LRRK2 that impact kinase function in one or both of the biochemical screens. Collectively, we observed that these two LRRK2 kinase events do not correlate with one another and have the potential to be uncoupled in the context of some LRRK2 coding variants. We also tested our novel SiMoA assays for use in a biomarker context by evaluating them across various biological sample matrices including rodent serum, and human cerebrospinal fluid and urine. The second proximity-labeling proteomics approach was adapted from a previous publication, modified for LRRK2 fusion and optimized for quantitative assessment of LRRK2 proximity network changes under various conditions of the enzyme. Herein we used the ascorbate peroxidase (APEX2) proximity labeling enzyme fused to our protein of interest, LRRK2. We performed appropriate quality control experiments and optimized the labeling and lysate processing approaches to improve the yield and reduce variability of the mass spectrometry results. We tested our APEX2-LRRK2 proximity-labeling fusion construct across a spectrum of LRRK2 kinase functional states: basal, pharmacological kinase inhibition and genetic activation states in vitro. We found that the active conformation of LRRK2 may be the most important in determining proximity networks independent of LRRK2s ability to phosphorylate. In all we developed a novel LRRK2 proximity-labeling tool optimized for quantitative experimental readouts. As LRRK2-targeted therapies enter efficacy stages in clinical trials, the identification of novel variant carriers that could benefit from LRRK2-targeted therapeutics combined with new efficacious biomarker strategies could be transformative to enhance precision approaches in genetically-defined disease. Moreover, the impacts of novel variants on kinase function combined with advanced proteomic evidence surrounding the molecular function of LRRK2 and disruptions that arise in disease states will be crucial in better understanding LRRK2’s typical function and role in disease pathogenesis.

Cellular senescence is a fundamental cell fate playing significant and complex roles during tumorigenesis and natural aging process. However, the molecular determinants distinguishing senescence from other temporary and permanent cell-cycle arrest states such as quiescence and post-mitotic state and the specified mechanisms underlying cell-fate decisions towards senescence versus cell death in response to cellular stress stimuli remain less understood. In our studies, we aimed to employ multi-omics approaches to deepen our understanding of cellular senescence, in particular, regarding the specific molecular determinants distinguishing cellular senescence from other non-dividing cell fates.

Notably, one of the most prominent features of cellular senescence differing from other non-dividing cell fates is the increased expression of senescence-associated beta-galactosidase. Because 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C12FDG) is known as the substrate catalyzed by beta-galactosidase for producing a green fluorescent product, we applied this compound to the cells undergoing G1 cell-cycle arrest (a mixture of senescent and quiescent cells). Employing fluorescence-activated cell sorting, we separated and collected senescent and quiescent cell populations based on green fluorescence intensity. As cellular senescence is more than just the non-dividing cell fate, we therefore systematically compared the gene expression between senescence and quiescence to provide insights into the specific features underlying senescence programming beyond cell cycle arrest. Following this strategy for the comparative gene expression analysis, we identified and characterized several genes critically involved in the program of cellular senescence, and one of the major findings was to identify IMMP2L, a nuclear-encoded mitochondrial intermembrane peptidase, can act as a molecular switch for determining the cell fates of healthy living, cell death, and senescence.

Inhibiting IMMP2L signaling through either the suicidal protease inhibitor SERPINB4 or transcriptional downregulation was sufficient to initiate cellular senescence by reprogramming the mitochondria functionality. Employing proteomics, we identified at least two mitochondrial target proteins processed by IMMP2L, including metabolic enzyme GPD2 and cell death regulator/electron transport chain complex I component AIF. Functional study suggests that, in healthy cells, the IMMP2L-GPD2 axis catalyzes redox reactions to produce phospholipid precursor Glycerol 3-phosphate; while under oxidative stress, IMMP2L cleaves AIF into its truncated pro-apoptotic form leading to cell death initiation to remove cells with irreparable damage. For cells programmed to senesce, the IMMP2L-GPD2 axis is switched off to block phospholipid biosynthesis leading to reduced availability of membrane building blocks for cell growth together with the disruption of mitochondrial localization of certain phospholipid-binding kinases, such as protein kinase C-δ (PKC-δ) and its downstream signaling. These alterations in mitochondria-associated metabolism and signaling network promote entry into a senescent state featuring high levels of reactive oxygen species (ROS). Simultaneously, blockage of pro-apoptotic AIF generation, which is due to the loss of IMMP2L, ensures the viability of senescent cells under ROS-mediated oxidative stress. Taken together, we have mechanistically uncovered IMMP2L-mediated signaling as a key regulatory pathway in the control of fates of healthy, apoptotic, and senescent cells.

In the physiological conditions, we observed that IMMP2L is downregulated in the muscle tissues and the blood samples of geriatric groups compared to that from young cohorts. Besides, centenarians display better genomic integrity at the IMMP2L locus when compared with the general population. Taken together, it suggests IMMP2L could also be an important player associated with the natural aging process.

The RAS family is a group of small GTPases that can become constitutively activated by point mutations that are found in about 30% of all cancer patients. There are three well-characterized RAS family members: HRAS, NRAS, and KRAS, the latter of which is alternatively spliced at the C-terminus into KRAS4A and KRAS4B. The RAS proteins are all nearly identical at their N-termini and core effector binding domains, but have divergent C-terminal membrane-binding regions that impart different subcellular localization and subtle differences in signaling. Although the role of constitutively activated oncogenic RAS has been well established to play a role in cancer, recent work has suggested that wild-type RAS signaling may also be important in tumorigenesis. Wild-type RAS proteins have been shown to be activated in the presence of oncogenic KRAS. However, the consequences of this activation are context-dependent, as signaling through the wild-type RAS proteins has been shown to both suppress neoplastic growth and promote tumorigenesis under different circumstances.

I sought to investigate the role of the wild-type RAS proteins in two clinically –relevant models of cancer: pancreatic, the type of cancer most frequently associated with KRAS mutations, and lung cancer, the cancer in which KRAS mutations affect the highest number of patients. First, I tested whether a loss of wild type Hras altered tumorigenesis in a mouse model of pancreatic cancer driven by oncogenic Kras. Hras homozygous null mice (Hras-/- ) exhibited more precancerous lesions of the pancreas as well as more off-target skin papillomas compared to their wild type counterparts, indicating that Hras suppresses early Kras-driven pancreatic tumorigenesis. Loss of Hras also reduced the survival of mice engineered to develop aggressive pancreatic cancer by the additional disruption of one allele of the tumor suppressor p53 (Trp53R172H/+). However, this survival advantage was lost when both alleles of Trp53 were mutated, suggesting that wild-type HRas inhibits tumorigenesis in a p53-dependant manner.

Next, I investigated the role that wild-type Hras and Nras play in a chemical carcinogen-induced model of lung cancer. In mice treated with urethane, a carcinogen that induces Kras-mutation positive lung lesions, Hras-/ mice once again developed more tumors than wild-type mice. Interestingly, however, this effect was not observed in mice lacking wild-type Nras. Mice lacking both Hras and Nras alleles developed approximately the same number of tumors as Hras-/- mice, thus the additional loss of Nras does not appear to enhance the tumor-promoting effects of loss of Hras. In summary, signaling through wild-type Hras, but not Nras, suppresses tumorigenesis in a carcinogen-induced model of lung cancer.

The tumor-suppressive effects of wild-type Ras signaling were traced to the earliest stages of pancreatic tumorigenesis, suggesting that wild-type Ras signaling may suppress tumorigenesis as early as the time of initiation. These findings suggest that differences in expression of the wild-type Ras isoforms could potentially play a role in an individual’s predisposition for developing cancer upon oncogenic insult.

The incidence of thyroid cancer, and in particular papillary thyroid cancer (PTC), is rising faster than that of any other malignancy in the United States. Thyroid cancer is now the most common endocrine cancer and the fifth most common cancer in women. While most thyroid cancers are treated effectively with surgical resection and radioiodine therapy, survival drops precipitously in metastatic or radioiodine-resistant disease. 60% of papillary thyroid cancers (PTC) have an oncogenic V600E mutation in the kinase BRAF, which leads to a constitutively active and oncogenic kinase. This mutation has been associated with as well as a 2.14-fold increase in recurrent/persistent disease. Excitingly, inhibitors against this mutant kinase or its substrates, the MEK1/2 kinases, can prolong progression free survival or stabilize disease in radioiodine-refractory thyroid cancer patients. However, the indolent nature of PTC may be a challenge to the clinical adaption of these inhibitors, as the financial and physical toxicities of these treatments may be amplified over the prolonged time-course typical of PTC. MEK1/2 require copper (Cu) for kinase activity and can be inhibited with the well-tolerated and economical Cu chelator tetrathiomolybdate (TM). Cu chelators have been in use for decades in patients with Wilson’s Disease, a disease of Cu accumulation. Unlike current BRAF and MEK inhibitors, Cu chelators can be well tolerated for decades with few side effects, and thus may find use in long-term inhibition of BRAFV600E signaling in PTC.

Here I test the ability of Cu chelator TM to inhibit tumor growth in BRAFV600E-mutant PTC. TM inhibited MEK1/2 kinase activity and transformed growth of human BRAFV600E-mutant PTC cells as well as or more potently than standard-of-care drugs. Consistent with TM deriving its antineoplastic activity by inhibiting MEK1/2, expression of activated ERK2, a substrate of MEK1/2, overcame the ability of TM to suppress growth of BRAFV600E-mutant PTC cells. TM was also effective in a genetically engineered mouse model of BrafV600E-mutant PTC; oral TM reduced tumor burden as well as a clinical BRAF inhibitor. This in vivo effect was attributed to a reduction of phospho-Erk1/2 signaling in the tumors. Additionally, long-term maintenance therapy using TM after cessation of a clinical BRAF inhibitor reduced tumor volume in the same mouse model. Genetic reduction of the Cu transporter CTR1 in developing tumors also trended towards a survival advantage in mice with BrafV600E-mutant PTC. Finally, TM also enhanced the antineoplastic activity of standard of care clinical BBRAF inhibitor drugs. These results support the clinical evaluation of Cu chelation as targeted therapy for BrafV600E-mutant PTC and suggests three possible avenues for clinical exploration: i) as a less toxic monotherapy for BrafV600E-mutant PTC, ii) as long-term maintenance therapy after initial treatment, and iii) as a combination-therapy amplifying the antineoplastic effects of other treatment modalities.

The gene BRAF is mutated to remain aberrantly activated in a large number of human malignancies, most prominently in melanoma. The most common mutation in BRAF is a missense mutation that substitutes glutamic acid for valine at codon 600 (V600E) that leads to constitutive activation of this kinase. In this active state, BRAFV600E phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate the ERK1 and ERK2 kinases of the MAPK pathway to promote tumorigenesis. Targeting this pathway is a well-validated strategy to treat BRAF-mutant cancer. Inhibitors of both BRAFV600E and the MEK1/2 kinases are used to treat BRAF-mutant melanoma and are being evaluated in other cancers as well. However, the duration of response to these targeted therapies is limited by innate and acquired resistance, which is often mediated through reactivation of the MAPK pathway. Thus, new targeted therapies to inhibit MAPK signaling in BRAF-mutant malignancies are required. To this end, MEK1/2 kinases require copper (Cu) for enzymatic activity and signaling. We therefore tested whether the dependency of these validated targets on Cu could be leveraged for the treatment of BRAF-mutant cancer.

We report that genetic reduction of Cu import through disruption of the gene encoding the high affinity Cu transporter CTR1 or pharmacological chelation of Cu with the drug tetrathiomolybdate (TTM) suppressed MAPK signaling in both in vitro and in vivo models of BRAF-mutant tumorigenesis. This reduction in MAPK signaling correlated with a reduced potential for tumorigenic growth and an increase in survival of tumor-bearing mice. Finally, TTM reduced the transformed growth of a number of human melanoma cell lines engineered to be resistant to current MAPK pathway inhibitors. As such, Cu chelation holds promise as a novel treatment for BRAF-mutant cancers and may find value in targeting resistance to current MAPK pathway inhibitors.

RAS is a small GTPase that helps to convert extracellular cues into intracellular actions. It is the most commonly mutated oncogene and is found in an active mutant form in 90% of pancreatic cancers. Therefore, study of how this protein is made and then how this protein signals in the cell could provide the foundation for novel approaches to treat RAS-driven malignancies.

First I demonstrate that the level of protein expressed from the gene KRAS, but not the highly homologous gene HRAS, is limited in mammalian cells by an abundance of underrepresented (rare) codons in the encoding mRNA. KRAS mRNA from both ectopic plasmids as well as from the endogenous cellular gene is subject to slowed translation due to these rare codons within its coding sequence. This has consequences for tumorigenesis, as replacement of the rare codons with commonly used codons accelerates RAS driven tumor growth. This may extend beyond HRAS and KRAS, as many other homologous gene pairs show a high divergence in codon usage and protein expression, suggesting that this could be a wider phenomenon used by mammalian cells to regulate protein levels.

Second, I demonstrate that RAS driven tumors partially depend on eNOS for growth. Using genetically engineered mouse models that recapitulate the spontaneous development of pancreatic cancer, I demonstrate that the protein eNOS is progressively upregulated as tumors develop. I then demonstrate that genetic ablation of eNOS partially blocks the development of preinvasive pancreatic lesions in these mice, and trends toward increasing survival in mice that develop lethal pancreatic adenocarcinoma. Furthermore, I then show that inhibition of eNOS using the clinically tested small molecule L-NAME can also slow the development of preinvasive neoplasia and nonsignificantly increase survival, although not to the level of eNOS genetic ablation. These findings are applicable to a clinical setting, as in conjunction with others I show that L-NAME treatment of human pancreatic cancer xenografts halves their growth, even when the main side effect of L-NAME, hypertension, is treated.

Together, these studies provide a better understanding of how RAS functions within the cell, and thus, ultimately, how RAS driven cancers may be treated.

The Ras proteins, composed of H, N, and KRas, are a family of small GTPases that normally transmit extracellular cues to the cell in a regulated manner. However, Ras is commonly mutated to be inappropriately activated in human cancers, promoting a vast array of tumor phenotypes. Activation of the Raf, PI3K, and RalGEF Ras effector pathways is required to promote Ras-mediated tumorigenesis, leading not only to cell autonomous tumor phenotypes, but also the establishment of a tumor microenvironment. However, following tumor initiation, the requirement upon oncogenic Ras signaling is reduced to activation of PI3K, most likely due to a contribution of the tumor microenvironment. In order to further delineate the requirements for oncogenic Ras signaling pathways during tumorigenesis, I sought to 1) identify PI3K-independent factors necessary for tumor initiation, and 2) determine how PI3K activation maintains tumor growth in the absence of oncogenic Ras. Using cell-based assays and tumorigenesis assays in mice, I have shown that interleukin-6 (IL-6) is secreted upon induction of Ras expression, is required for Ras-mediated tumor initiation, and promotes tumorigenesis in a paracrine manner by fostering angiogenesis. Additionally, I have shown that eNOS, a downstream target of the PI3K pathway, is required for Ras-induced tumor initiation and maintenance, and, moreover, that eNOS-mediated S-nitrosylation and activation of wildtype Ras proteins is required throughout tumorigenesis. Pancreatic cancer is the cancer most highly associated with oncogenic Ras mutations, and I have shown that both IL-6 and eNOS are required for the tumorigenic growth of pancreatic cancer cell lines in mice. I therefore suggest that these proteins, perhaps in combination with other Ras inhibitors, may provide potential anti-cancer targets for oncogenic-Ras driven cancers in the clinic.

Epithelial cells are the organism’s first defensive barrier to pathogens, toxins, and other noxious elements. Constant exposure to these elements requires exquisite control over the process of regeneration to ensure that damaged cells are constantly replenished while preventing overgrowth and maintaining organ size. Many of the mechanisms of proliferation and differentiation are exploited by sub-populations of cells following injury but also by tumor cells to allow uncontrolled growth and acquired resistance.

In the lung, elegant studies have identified several putative stem cell populations contributing to the regeneration of lung epithelial cells following injury. However, despite the abundance of region specific lung stem/progenitor cells, a specific cellular source that can be activated in response to damage remains unknown. The studies presented here identify the Abl kinases as a potential cellular target in several progenitor cell types that can be targeted for efficient recovery following a variety lung injuries, including bacterial and viral pneumonia. Furthermore, by targeting some of the same pathways of de-differentiation cancer cells use to acquire resistance, Abl kinase inhibition promotes differentiation of primary lung cancer cells and renders them more susceptible to chemotherapy.

The genes encoding the Ras family of small GTPases are mutated to yield constitutively active GTP-bound oncoproteins in one-third of all human cancers. In many other cancers lacking Ras mutations, Ras is activated by other means. One common example of such activation is found in breast cancer, in which epidermal growth factor receptor (EGFR) family receptor tyrosine kinases, including EGFR and HER2 (ErbB-2/Neu), are frequently amplified and overexpressed, which in turn activates Ras. In human cells, activation of the Ral guanine nucleotide exchange factor, or RalGEF, effector pathway is necessary for Ras-mediated tumorigenesis and metastasis. RalGEFs activate the two highly similar Ral GTPases, RalA and RalB. While RalA has been shown to be required for Ras-mediated tumorigenesis, RalB is important for tumor metastasis. Activated Ral GTPases bind to and activate a limited number of effector proteins, including RalBP1, Sec5, and Exo84, to affect numerous diverse activities of the cell. This dissertation research sought to determine which of these well-characterized Ral effector proteins were required for oncogenic mutant Ras-induceded tumorigenesis and metastasis of human cells, as well as to examine the role of RalA in breast cancer cells that can activate Ras through EGFR and HER2 overexpression.

RNA interference-mediated loss-of-function analysis demonstrated that Sec5 and Exo84 are required for oncogenic Ras-mediated tumorigenesis, and, at least in part, metastasis. Additionally, both gain-of-function and inhibition studies showed that RalA activation is induced by EGFR and HER2 in breast cancer cell lines stimulated with EGF. Furthermore, stable suppression of RalA expression inhibited tumorigenic growth of breast cancer cells, and RalA activation was shown to be higher in a majority of mammary adenocarcinomas versus matched patient normal mammary tissue. These studies provide new insights into the importance of RalA activation in breast cancer, as well as the molecules downstream of RalA and RalB that may be responsible for mediating their effects on tumorigenesis and metastasis.

The Ras family of small GTPases, comprised of the KRAS, NRAS, and HRAS genes, are mutated to encode constitutively-active, GTP-bound, oncogenic proteins in upwards of one quarter or more of all human cancers, which is well established to promote tumorigenesis. Despite the prominent role these genes play in human cancer, the encoded proteins have proven difficult to pharmacologically inhibit. Therefore, it is important to understand how Ras proteins are activated.

RAS proteins cycle between a GDP-bound inactive state and a GTP-bound active state through guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). GEFs facilitate the GDP-to-GTP exchange of RAS and promote RAS activation. Similar to GEFs, reactive oxygen/nitrogen species can also promote RAS activation through reactions with the thiol residue of cysteine 118 (C118). This residue may therefore play a role in RAS activation in cancer. To test this possibility, I investigated the effect of mutating C118 to serine (C118S) in Kras on (1) carcinogen-induced lung tumorigenesis, and (2) xenograft tumor growth of HRAS12V-transformed cells.

To explore the impact of the C118S mutation in Kras on carcinogen-induced lung tumorigenesis, I introduced a C118S mutation into the endogenous murine Kras allele and exposed the resultant mice to the carcinogen urethane, which induces Kras mutation-positive lung tumors. Kras+/C118S and KrasC118S/C118S mice developed fewer and smaller lung tumors than Kras+/+ mice. Although the KrasC118S allele did not appear to affect tumorigenesis when the remaining Kras allele was conditionally oncogenic (KrasG12D), there was a moderate imbalance of oncogenic mutations favoring the native Kras allele in tumors from Kras+/C118S mice treated with urethane. Therefore, mutating C118 of Kras impedes urethane-induced lung tumorigenesis.

To explore the the impact of the C118S mutation in Kras on xenograft tumor growth of HRAS12V-transformed cells, I tested and found that redox-dependent reactions with cysteine 118 (C118) and activation of wild type KRAS are critical for oncogenic HRAS-driven tumorigenesis. Such redox-dependent activation of KRAS affected both PI3K-AKT and RAF-MEK-ERK pathways. These findings were confirmed in the endogenous mouse Kras gene. Speicfically, oncogenic HRAS-transformed KrasC118S/C118S MEFs grew in soft agar and as xenograft tumors more slowly than similarly transformed Kras+/+ MEFs, suggesting that redox-dependent reactions with C118 of Kras promotes transformation and tumorigenesis.

Taken together, I have demonstrated a critical role of redox-dependent reactions with Kras C118 in tumorigenesis.