CANCER BIOLOGY Roles of Fibroblast MMP2 in Breast to Lung Metastasis Andreia L. Bates Dissertation under the direction of Barbara Fingleton, PhD Breast cancer five-year survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. The potential for tumor cells to proliferate in a foreign microenvironment and develop into overt metastases is mediated through interactions with stromal cells at the secondary site. Understanding the mechanisms underlying tumor cell cooperation with the stromal milieu to support the outgrowth of metastases is essential for increasing patient survival. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. Preliminary studies found that loss of host MMP2 reduced the proliferation of experimental metastases in the lungs of mice, and identified fibroblasts in control tumor-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumor growth was increased by co-culture with control fibroblasts isolated from tumor-bearing lungs but not when fibroblasts with Mmp2 knockdown were used. This result suggested that MMP2 was responsible for a tumor-proliferative, activated fibroblast phenotype. The studies within this dissertation employ a combination of in vitro, in vivo, and correlation analyses using a human data set to investigate how MMP2 potentiates breast tumor outgrowth in the lungs. In vitro, exogenous active enzyme increased tumor cell proliferation in 3D but not 2D. Knockdown of Mmp2 in fibroblasts attenuated expression of two markers of activation (á-smooth muscle actin and vimentin). Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Active TGFâ-1 was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked with addition of TGFâ-1 neutralizing antibody. Gene expression data in stromal cells of human breast cancers revealed that MMP2 expression is also positively correlated with activation and matrix transcripts. A model is presented whereby MMP2 production in tumor fibroblasts is important for the activity of TGFâ-1 cytokine and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, these studies reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extends the mechanisms by which MMPs contribute to tumor progression. Approved___________________________ Date_______ Barbara Fingleton, PhD

Melanoma is the deadliest form of skin cancer, and virtually all patients progress on targeted therapies. Dysregulated metabolism has been shown to affect therapy response, so BRAF-mutated melanoma cell line models were used to connect cellular metabolism to therapeutic proliferative response. The data show that forcing a glycolytic metabolic strategy in the context of drug treatment enhances the antitumor effect. Anti-retrovirals, particularly zalcitabine, were shown to dramatically affect proliferation when combined with BRAF inhibitor. All in all, this dissertation provides an important contribution to response variability and assay development, the glycolytic biology in relation to BRAF inhibition, and a finer inspection of variability within a tumor.

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Le cancer bronchique reste la première cause de mortalité par cancer en France (21 000 décès chez l’homme et 7700 chez la femme, Institut national de veille sanitaire, 2010). Il semble alors essentiel, outre les soins médicaux, de favoriser et de préserver la qualité de la relation entre le personnel soignant, les médecins et les patients. Une des composantes essentielles dans la prise en charge des patients est l’empathie. Afin de pouvoir travailler l’aspect empathique au sein de la relation, encore faut-il parvenir à cerner les attentes des patients et comment ils perçoivent les aptitudes empathiques des soignants et des médecins à leur égard. C’est dans cette perspective que nous avons élaboré une échelle d’empathie perçue et mené des entretiens avec des patients. Nous avons créé cette échelle à partir de la littérature et des échelles existantes. Cette échelle comporte deux volets : un « équipe soignante » et un « équipe médicale ». Nous l’avons soumise à 112 patients pour la version « équipe soignante » et 116 patients pour la version « équipe médicale ». Des éléments de validation statistique témoignent de leurs qualités en termes de fiabilité et de structure factorielle. Les entretiens menés parallèlement révèlent que les patients attendent des qualités empathiques distinctes de la part des médecins ou des infirmières. Ce que l’on retrouve dans la littérature. Ces attentes spécifiques sont en lien avec les représentations que les patients ont de la profession considérée. Ces résultats devront permettre de proposer ultérieurement une formation aux équipes soignantes et médicales afin de favoriser l’établissement d’une relation empathique avec le patient. / Lung cancer is the first cause of death by cancer in France. An important component of care relationship between nurses/physicians and patients is empathy. To study empathy in this context, we analyzed how patients perceive nurses and physicians empathy qualities. To consider this aspect, we constructed a “Perceived Empathy Scale of Physicians and Nurses”, and we conducted interviews with lung cancer patients. This scale is composed by two parts: one for the “nurse’s team” and the other for the “physician’s team” (both evaluated by patients). 112 patients completed the first scale’s part “nurse’s team” and 116 patients the second scale’s part “physician’s team”. Many statistic validation factors proved that the instrument had good psychometrics qualities. Interviews with patients revealed that they expected different empathic qualities from nurse and from physician. These results are linked to patient’s professional (nurse & physician) representations. Our future objective is to provide physicians and nurses an “empathy training program”, in order to improve empathic relationship between cancer patients and health professionals.

It was been well established that the Activin A signaling pathway plays a pivotal role in the developing, adult, and diseased organism, commonly acting as a growth inhibitor. Though prevalent in embryonic tissues, Activin A secretion is downregulated in post-natal tissues. Contrary to its function, Activin A expression is often upregulated in cancer, and, of particular interest, in esophageal (ESCC) and head and neck squamous (HNSCC) cell carcinomas. Therefore, we investigated the function of Activin A in these contexts. Interestingly, we found that Activin A acts as an inhibitor of invasion and regulator of the extracellular matrix on dysplastic and ESCC cell lines that retain expression of Activin A receptor type IB, ACVRIB. When ACVRIB is lost, Activin A no longer exerts these effects, though the other components of the Activin A receptor complex remained intact. Consequently, we decided to further define the functional effects of loss of ACVRIB in HNSCC and ESCC cells using CRISPR/Cas9 and siRNA, respectively. In the absence of ACVRIB, we found increased proliferation, migration, and invasion. Using an organoptypic culture system and immunofluorescence staining, we observed altered expression of proteins responsible for cell-cell and cell-extracellular matrix adhesion. We conclude that ACVRIB-dependent Activin A signaling is necessary to regulate ESCC and HNSCC progression.

Despite recent progress, non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality in the United States and new therapeutic approaches are needed. Nuclear factor κB (NF-κB) is a master regulator of inflammatory signaling that is overexpressed in most solid tumors. Several mouse models of lung cancer have confirmed the requirement for NF-κB signaling in airway epithelial cells during lung tumorigenesis. However, despite these findings, inhibitors of NF-κB have been ineffective in treating NSCLC patients. Using genetic and pharmacologic inhibition of NF-κB signaling in murine lung cancer models, we found that blockade of NF-κB signaling in the myeloid inflammatory cell population paradoxically increases lung inflammation, airway epithelial cell proliferation, and lung tumorigenesis. We identified cathepsin G-mediated processing of IL-1β by neutrophils as a novel resistance mechanism of NSCLC to NF-κB inhibitors, and combined therapy with an NF-κB inhibitor and IL-1 receptor antagonist reduced tumorigenesis in mouse models of lung cancer. In NSCLC patients, plasma IL-1β concentration inversely correlated with progression-free survival and IL-1β levels were increased following treatment with an NF-κB inhibitor. These studies demonstrate that targeting common signaling pathways can have opposing effects in individual cell types during tumorigenesis; they support the use of rational, combined therapies to treat lung cancer.

Head and neck squamous cell carcinoma is a frequent and serious malignancy that accounts for more than 300,000 deaths world wide each year, the majority of which are the result of local invasion and lymph node metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), transcriptional adaptors that have important roles in normal epithelial cell differentiation, have also been associated with increased lymph node metastasis and decreased differentiation in carcinomas of the breast and of the head and neck. Here within, LMO4 and LDB1, in addition to single-stranded-binding proteins (SSBPs) are involved in regulating invasion, and provides an explanation for why LMO4 and LDB1 have concordant levels of expression in this disease. This work directly shows that VU-SCC-1729 cells can invade cellular monolayers and that loss of LDB1 in these cells not only reduces invasion, but also plays an unexpected role in the reduction of proliferation. A loss of LDB1 in VU-SCC-1729 cells also resulted in reduced tumor volume and vascularization in a xenograft tumor model. Genome-wide DNA occupancy studies evaluated LDB1 and SSBP2 binding patterns in head and neck cancer cells. Together, these data suggest that LMO4, LDB1, and SSBPs may have a collective role in regulating processes critical for progression of head and neck carcinoma including invasion, proliferation, and angiogenesis.

In spite of recent advances in therapy, cancer remains a leading cause of death worldwide. Therapy response is often unpredictable and relapse frequently occurs. In many cases, this therapy resistance is attributed to subsets of therapy resistant cancer cells and surrounding stromal cells that support a resistant phenotype. A better understanding of cellular heterogeneity in cancer is therefore crucial in order to develop novel therapeutic strategies and improve patient outcomes. Experimental technologies like mass cytometry (CyTOF) allow for high-content, multi-parametric single-cell analysis of human tumor samples. However, analytical tools and workflows are still needed to standardize and automate the process of identifying and quantitatively describing cell populations in the resulting data. This dissertation presents a novel workflow for automated discovery and characterization of novel and rare cell subsets, quantification of cellular heterogeneity, and characterization of cells based on population-specific feature enrichment. First, a modular workflow is described that combines biaxial gating, dimensionality reduction, clustering, and hierarchically clustered heatmaps to maximize rare population discovery and to create an interpretable visualization of cell population characteristics. Next, a novel method is introduced for quantifying cellular heterogeneity based on two-dimensional mapping of cells in phenotypic space using tSNE analysis. Finally, an algorithmic method termed Marker Enrichment Modeling (MEM) is introduced that automatically quantifies population-specific feature enrichment and generates descriptive labels for cell populations based on their feature enrichment scores. MEM analysis is shown to identify features important to cell identity across multiple datasets, and MEM labels are effectively used to compare populations of cells across tissue types, experiments, institutions, and platforms. Going forward, the tools presented here lay the groundwork for novel computational methods for machine learning of cell identity and registering cell populations across studies or clinical endpoints. Automated methods for identifying and describing cell populations will enable rapid discovery of biologically and clinically relevant cells and contribute to the development of novel diagnostic, prognostic, and therapeutic approaches to cancer and other diseases.

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer worldwide. OSCC commonly invades into the lymph nodes and mandible, which correlate with increased rates of recurrence and lower overall survival. Tumors that infiltrate mandibular bone proliferate rapidly, cause large amounts of bone destruction and require extensive surgeries. Unfortunately, the molecular mechanisms of OSCC invasion into the mandible are not well understood and survival rates have not significantly improved for over 30 years. In a syngeneic model of murine OSCC, Parathyroid Hormone-related Protein (PTHrP), has been shown to be required for OSCC invasion into the mandible. Existing studies have identified the Hedgehog (Hh) transcription factor, Gli2 as the regulator of PTHrP and our previous work in breast cancer metastasis to bone suggest that TGFB may regulate Gli2 transcription. Here we demonstrate that Hh and TGFB signaling concomitantly regulate Gli2 and subsequently PTHrP in OSCC. Additionally, we have elucidated a mechanical signaling mechanism that controls Gli2 activation levels, where extra-cellular matrix rigidities similar to bone, but not basement membrane, causes an increase in ciliogenesis. We also demonstrate the feasibility of targeting Gli2 in vivo using GANT58 loaded microspheres to prevent bone destruction.

Lung cancer is the leading cause of cancer deaths worldwide. More than 40% of lung cancers are classified as lung adenocarcinoma (LUAD), which is defined by pathological characteristics. Oncogenic mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain (TKD) are found in a significant portion of LUAD. Such mutations, which present most commonly as small in-frame deletions in exon 19 (ex19del) or as point mutations in exon 21 (L858R), allow for EGFR activation, sensitivity to EGFR tyrosine kinase inhibitors (TKIs), and molecularly targeted therapy in this malignancy. By analyzing the tumors of patients with LUAD, we identified two EGFR alterations that had not previously been reported in lung cancer: the EGFR kinase domain duplication (EGFR-KDD) and the EGFR-RAD51 fusion. While knowledge about common EGFR mutations (L858R or ex19del) has allowed for rational treatment of specific cohorts of LUAD, nothing was known about the EGFR-KDD and EGFR-RAD51 in LUAD. These mutationsâwhich we call EGFR rearrangementsâmaintain the wild type sequence of the EGFR TKD and could have mediated either sensitivity or primary resistance to EGFR-TKI therapy. The goal of these studies was to fill in this knowledge gap: to characterize a new type of EGFR alteration in LUAD, to determine the sensitivity of these rearrangements to therapy, and, along the way, to uncover more about the biology of EGFR. Collectively, the research in this dissertation used in vitro and in silico models, as well as patient-derived data, to address a previously unrecognized potential mechanism of oncogenesis in lung cancer: EGFR rearrangements. These studies established EGFR-KDD and EGFR-RAD51 as bona fide recurrent oncogenes and therapeutic targets in LUAD. The work is already proving clinically relevant as these alterations have been identified in additional lung cancer patientsâwho may now have more targeted treatment options.