Browsing by Subject "Cancer biology"

The APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of cytidine deaminases is one of the most common endogenous sources of single base substitution mutations in human cancer. Accordingly, APOBEC enzymes represent a major source of intratumor genetic heterogeneity and have been associated with immunotherapy response in diverse cancer types. However, the consequences of APOBEC mutagenesis on tumor progression in vivo are not well understood. To address this, I developed several murine tumor models with inducible APOBEC3B expression and studied the contribution of APOBEC activity to tumor evolution and immunogenicity. First, I explored the effects of APOBEC activity on tumor relapse using a murine model of mammary tumor recurrence. APOBEC activity led to a significant acceleration in tumor recurrence following the strong selective pressure of oncogenic driver signaling loss. Recurrent APOBEC tumors had undifferentiated histological features and large, irregularly shaped nuclei containing defects like micronuclei, multinucleation, and chromatin bridges. I found that recurrent APOBEC tumors amplified the therapy resistance-associated oncogene, c-Met, on circular extrachromosomal DNA, likely driving the proliferation of the recurrent cancer cells. Second, because APOBEC mutational signatures are enriched in the majority of HER2-positive breast cancer patients, I used a syngeneic HER2-driven mammary tumor model to study the effects of APOBEC activity on the tumor immune microenvironment. I found that APOBEC activity induced an antitumor adaptive immune response and CD4+ T cell-mediated tumor growth inhibition. While polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected by the immune system, suggesting that APOBEC-mediated genetic heterogeneity limits the antitumor adaptive immune response. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. Consistent with the observed immune infiltration in murine APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to checkpoint inhibition. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures and clonality as biomarkers predicting immunotherapy response in HER2-positive breast cancers. In conclusion, I’ve identified a novel role for APOBEC activity in generating chromosomal instability, consisting of mitotic errors, oncogene amplification, and extrachromosomal DNA formation to promote tumor recurrence. Moreover, APOBEC activity also stimulated an antitumor adaptive immune response and sensitized tumors to immunotherapy.

AbstractFor over 30 years the cancer biology field has scrutinized the mechanisms behind p53 and RB mediated tumor suppression (Fields and Jang 1990; Kern et al. 1991; Raycroft, Wu, and Lozano 1990; Dyson 1998; Classon and Harlow 2002; Knudsen and Knudsen 2008) Together, these genes regulate complex interconnecting pathways responsible for the regulation of cell growth, cell death, and genomic integrity (Sherr and McCormick 2002). Not surprisingly, the pathways regulated by these two tumor suppressors are almost universally disrupted during the development of cancer (Hanahan and Weinberg 2000). By nature, tumor suppressors play a key role in preserving the integrity of the genome. Whether in response to injury or in maintaining stable gene expression patterns, p53 and RB have been well established as two of the most important factors preventing genomic instability, epigenetic deregulation, transformation, and tumorigenesis. However, the precise mechanism by which p53 or RB mediate tumor suppression remains unclear (Tiwari, Jones, and Abrams 2018). Over the last decade, several studies in genetically engineered mouse models have demonstrated that the canonical functions of these two tumor suppressors fail to fully explain their tumor suppressive capabilities (Mello and Attardi 2018; Janic et al. 2018; T. Li et al. 2012). Moreover, expanding literature continues to highlight a strong correlation between transposable element de-repression and several types of cancer, however, no definitive link between transposable elements and tumorigenesis has been established (Tiwari, Jones, and Abrams 2018; Wylie et al. 2016; Tiwari et al. 2020; Rodriguez-Martin et al. 2020). At present, the role that genomic repetitive elements play in tumorigenesis remains an open question. Here, to elucidate a possible mechanism of transformation we sought to investigate the link between the concurrent loss of p53 and RB and the de-repression of transposons. While individually, p53 and RB have been implicated in separate transposable element defense mechanisms, each responsible for preventing the expression of transposons (Ishak et al. 2016; Wylie et al. 2016; Tiwari et al. 2020; Dick et al. 2018; Tiwari, Jones, and Abrams 2018), my thesis work seeks to understand the role these repetitive elements play in transformation. Specifically, I investigate how the combined loss of p53 and RB affect the expression of transposons and how this relates to the development of cancer. Using genetically engineered mouse models we derived several lines of mouse embryonic fibroblasts (MEFs) which, through cre-lox technology, allowed for the deletion of floxed tumor suppressors (H. Kim et al. 2018). We derived MEFs from mice containing floxed p53, RB, both p53 and RB as well as several p53 and RB mutants. Generating primary MEFs allowed us to interrogate the loss of p53 and RB in a non-cancerous context, free of additional mutations (Todaro and Green 1963; Xu 2005). Moreover, by choosing MEFs as a model system, we could harness and observe the transformation process in a minimally manipulated system. Accordingly, we transformed normal Wild Type (WT) MEF cells and induced transformation by recombining loxP sites flanking p53 and RB. In dissecting whether loss of p53 and RB affected transposable element expression we showed that loss of p53 or RB alone each partially derepress long interspersed nuclear element 1 (LINE1) transposable elements, but remarkably co-deletion of p53 and RB together not only transform cells but also simultaneously induce massive expression of LINE1. Additionally, through the use of p53 transactivation domain mutants, we showed that the ability of p53 to repress transposable elements is a non-canonical function linked to tumor suppression. Interrogation of short interspersed nuclear elements (SINEs) produced similar findings. Furthermore, the derepression of both LINEs and SINEs appears to be regulated by the modification of the H3K9me3 histone mark. These results further correlate the expression of transposable elements to transformation and tumorigenesis. Complementary work revealed that loss of both p53 and RB in MEFs not only derepressed transposable elements and transformed cells, but in the process, massively rearranged the three-dimensional (3-D) chromatin landscape. This change in 3-D chromatin architecture resulted in the loss of intrachromosomal loops and the subsequent overexpression of several oncogenes. To assess the scope of the rearrangement of chromatin architecture, we developed a UCSC genome browser based atlas, mapping thousands DNA loops stemming from promoters, enhancers and silencers. We find that while the general structure of topologically associated domains are largely stable (Dixon et al. 2012), following deletion of p53 or RB, local chromatin contacts are vastly reorganized. Moreover, the reorganization of DNA loops was found to affect gene expression in a context dependent manner. These results support a model of p53 and RB as guardians of the genome that cooperate to maintain the chromatin architecture required for normal cellular function and prevent changes in DNA topology that promote uncontrolled growth associated with cellular transformation. More than half of all cancer patients have mutations in p53 or RB and many harbor evidence of deregulated transposable elements amidst a disordered chromatin landscape. Here, we explore the distinct and overlapping genomic regulatory processes that p53 and RB cooperatively maintain to preserve genomic integrity and prevent cancer. Overall, my work implicates transposable element derepression as a mechanism that can promote the development of cancer and moreover, provides a genome wide atlas of how p53 and RB can affect the chromatin landscape to regulate gene expression in a context dependent manner. Moreover, my work provides mechanistic insight into the tissue specific functions of p53 and RB. Taken together, this thesis provides new insights into the mechanisms by which p53 and RB prevent cancer.

Cancers are a complex set of related diseases with wide ranging etiologies, and humans are exposed to a milieu of environmental exposures that may contribute to disease development. Breast cancer in particular is a complex and multifactorial disease, yet prior research has largely focused on studying exposures to one factor/contaminant at a time, which does not reflect the real-world environment. In addition, investigating incidence of total breast cancer has the potential to mask the impacts of environmental factors on 1) the development of different subsets of breast cancer, varying in hormone receptor status and 2) progression of these cancers to advanced and more aggressive stages. Patients with distant metastatic breast cancer have poor prognosis despite aggressive, multidisciplinary treatment regimens compared to carcinoma in situ or early-stage breast cancer. This reinforces the unmet need to identify risk factors associated with advanced breast cancers to reduce incidence and improve overall breast cancer outcomes. The objective of these studies was thus to determine environmental risk factor associations with breast cancer by stage, and to utilize a real world environmental exposure chemical mixture to interrogate potential impacts on breast tumor progression by hormone receptor status.

These studies begin by evaluating the associations between breast cancer summary stages and the Environmental Quality Index (EQI), which includes a range of environmental factors across five overarching environmental domains. Our analysis found total and localized breast cancer incidence were increased in counties with poor environmental quality compared to those with good environmental quality. These overall environmental trends were largely driven by land quality, which was associated with incidence of early-stage disease (carcinoma in situ and localized breast cancer), in addition to total breast cancer, especially in urban counties.

Taking advantage of more granular patient data, these studies further evaluated the odds of localized, regional, or distant metastatic breast cancer in categories of environmental quality using women with carcinoma in situ as registry-based controls. Overall environmental quality was not associated with invasive breast cancer; however, poor land environmental quality was associated with increased odds of all invasive breast cancer types, particularly in more rural communities (distant metastatic breast cancer was 5-8% more likely compared to carcinoma in situ). Cumulatively, epidemiologic analyses indicate significant associations between poor overall and land environmental quality and breast cancer incidence as well as odds of invasive breast cancer. Furthermore, associations differed by breast cancer summary stage, rural-urban status, and environmental domain thus identifying a critical need to assess cumulative environmental exposures in the context of cancer stage.

Emerging evidence suggests the role of environmental chemicals, in particular endocrine disrupting chemicals (EDCs), in incidence and progression of breast cancer, which can impact survival outcomes. Polycyclic aromatic hydrocarbons (PAHs) are a toxic and ubiquitous class of environmental chemicals, many of which exhibit endocrine disrupting activity. They are products of fuel combustion from human and natural sources. PAH exposure is widespread, and many PAHs are considered carcinogenic. Urinary metabolite data were collected from 9517 individuals from the U.S. CDC National Health and Nutrition Examination Survey years 2005-2014 for four parental PAHs naphthalene, fluorene, phenanthrene, and pyrene. We utilized these urinary biomarkers to estimate PAH intake, and regression models were fit for multiple demographic and lifestyle variables, to determine variable effects, interactions, odds of high versus low PAH intake. Smoking and secondhand smoke (SHS) exposure accounted for the largest PAH intake rate variability (25.62%), and there were strongest interactions between race and ethnicity and smoking or secondhand smoke exposure, reflected in a much greater contribution of smoking to PAH intake in non-Hispanic Whites as compared to other racial and ethnic groups. Increased risk of high PAH intake was observed in older age groups, obese persons, college graduates, midrange incomes, smokers, and those who were SHS exposed. Among the non-smoking population, effects of other demographic factors lessened, suggesting a highly interactive nature. Results suggest that there are demographic subpopulations with high PAH intake because of different smoking behaviors and other exposures.

The PAH-contaminated Atlantic Wood Industries, Inc. (AWI) Superfund site in Portsmouth, Virginia provides a model for studying a real-world complex PAH mixture, and its extrapolation to cancer risk and PAH exposure in the general population. Cancer risk at the Superfund site due to sediment-derived PAHs was examined and PAH sources in the general population upon PAH mixture exposure were then evaluated. The PAH mixture was assessed for potential carcinogenicity using US EPA’s OncoLogic ranking tool and the US EPA list of priority PAHs. Cancer risk due to PAH exposure was calculated for Superfund site users and compared to the US EPA assessment. Human intake and health endpoints of PAHs within the mixture were extracted from USEtox chemical fate database. Eleven PAH compounds within the mixture were of carcinogenic concern, and seven PAHs conveyed significant excess cancer risk at the Superfund site and in the general population, wherein PAH-contaminated seafood ingestion was a main route of exposure.

Most research tends to focus on tumor etiology and the effect of single chemicals, offering little insight into the effects of realistic complex mixture exposures on tumor progression. To address this issue, the final portion of this work investigated the effect of the previously mentioned AWI Superfund site-derived PAH-enriched EDC mixture in a panel of normal mammary epithelial cells and breast cancer cell models. Cells or organoids in culture were treated with EDC mixture at doses estimated from U.S. adult intake of the top four PAH compounds within the mixture from the National Health and Nutrition Examination Survey database. Physiologic doses of this PAH mixture (6, 30, 300nM) demonstrated increased aryl hydrocarbon receptor (AhR) expression and CYP activity in estrogen receptor (ER) positive, but not normal mammary epithelial cells or ER negative breast cancer cells. In addition, upregulated AhR signaling corresponded with increased cell proliferation and expression of anti-apoptotic and antioxidant proteins XIAP and SOD1. A mathematical model was employed to validate PAH-mediated increases in AhR and XIAP expression in the MCF-7 ER-positive cell line. Furthermore, the PAH mixture caused significant growth increases in ER-negative breast cancer cell derived 3D tumor organoids, providing further evidence for the role of a real world-derived PAH mixture in enhancing a tumor proliferative phenotype.

Results from these studies have implications for cancer control and prevention and targeted PAH exposure reduction initiatives by identifying vulnerable subpopulations, and further identify key consequences of PAH exposure and how it may contribute to breast cancer biology and progression.

Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer. Although some TN breast cancers respond initially to neoadjuvant chemotherapy, the majority of patients die within three years of treatment due to recurrent tumor growth. Developing ex vivo models for TN breast cancer recurrence and defining responsible molecules will be crucial to developing effective combination therapies for TN breast cancer patients. We have developed an in vitro model of TN breast cancer dormancy/recurrence. Short-term exposure of tumor cells to chemotherapy at clinically relevant doses enriches for a dormant tumor cell population. Several days after removing chemotherapy, dormant tumor cells regain proliferative ability and establish colonies, resembling tumor recurrence. Tumor cells from "recurrent" colonies exhibit increased chemotherapy resistance, resembling therapy resistance of recurrent tumors in patients. Furthermore, we identify a novel signaling axis [nuclear bFGF/DNA-dependent protein kinase (DNA-PK)] supported by chemotherapy-enriched dormant TN breast cancer cells. This signaling axis drives accelerated DNA repair in chemo-residual TN breast cancer cells. Targeting this axis with either with a bFGF shRNA or DNA-PK small molecule inhibitor blocks recurrent colony formation. Using the Oncomine gene expression database, we found that bFGF expression in tumor samples from TN breast cancer patients predicts five year tumor recurrence following neoadjuvant chemotherapy treatment. Finally, we demonstrate that recurrent tumor cells exhibit increased invasiveness, reflecting the aggressive behavior of recurrent tumors in patients. Collectively, these studies identify a novel signaling axis in TN breast cancer that likely contributes to tumor recurrence and provide molecular targets for developing future therapeutics against TN breast cancer.