SH3PXD2A (SH3 and PX domains 2A)

The TKS5 protein, encoded by the gene SH3PXD2A, is a scaffolding protein essential for the formation of podosomes and invadopodia in untransformed cells and cancer cells, respectively. Podosomes and invadopodia (which collectively are termed invadosomes) are actin-rich cellular protrusions capable of secreting proteolytic enzymes that can degrade the extracellular matrix. These structures are thought to regulate cellular migration and invasion, as well as adhesion and the release of growth factors. In the context of cancer, TKS5-dependent invadopodia activity has been shown to play important roles in tumor growth and metastasis in various cancer types. Multiple isoforms of TKS5 exist due to alternative mRNA splicing and promoter usage

SH3PXD2A (SH3 and PX domains 2A)

The TKS5 protein, encoded by the gene SH3PXD2A, is a scaffolding protein essential for the formation of podosomes and invadopodia in untransformed cells and cancer cells, respectively. Podosomes and invadopodia (which collectively are termed invadosomes) are actin-rich cellular protrusions capable of secreting proteolytic enzymes that can degrade the extracellular matrix. These structures are thought to regulate cellular migration and invasion, as well as adhesion and the release of growth factors. In the context of cancer, TKS5-dependent invadopodia activity has been shown to play important roles in tumor growth and metastasis in various cancer types. Multiple isoforms of TKS5 exist due to alternative mRNA splicing and promoter usage

Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival

The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients

Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk

Transcriptional regulation of metastatic progression in lung adenocarcinoma

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.Lung cancer is the most prevalent cancer type, leading to more than one million deaths per year worldwide. The vast majority of these mortalities were attributed to metastasis, which is the dissemination of tumor cells from the lungs to other organs. The molecular mechanisms for metastasis is complex and not well understood. In this thesis, I investigated the gene expression changes in tumor cells that contribute to metastasis of lung adenocarcinoma, the major subtype of lung cancer. Using a genetically-engineered mouse model and derivative cell lines, we showed that metastatic lung adenocarcinoma cells are capable of forming proteolytic membrane protrusions known as invadopodia to degrade the extracellular matrix. The formation and function of invadopodia are dependent on an isoform switch of the adaptor protein Tks5. The Tks5long isoform, which is upregulated in metastatic cells, is capable of localizing to the cell membrane and activating invadopodia formation. In contrast, the Tks5short isoform, which is transcribed from a promoter independent of Tks5long, is the predominant isoform in non-metastatic cells, and functions to inhibit invadopodia-mediated matrix degradation by destabilizing these protrusions. We demonstrated that an increased ratio of Tks5long-to- Tks5short promoted invadopodia activity in vitro and metastasis in vivo. Furthermore, a high Tks5long-to-Tks5short ratio in human tumors correlated with advanced stage and worse survival. These data strongly suggest that a balance between Tks5long and Tks5short expression is critical for metastasis. In addition, we found that the expression of the pro-metastatic Tks5long isoform is synergistically inhibited by three transcription factors - Nkx2-1, Foxa2, and Cdx2. These three factors were highly expressed in non-metastatic cells, and downregulated in metastatic cells. Altered expression of these factors led to commensurate changes in Tks5long levels. Finally, we demonstrated that Nkx2-1, Foxa2, and Cdx2 function cooperatively to inhibit metastasis by suppressing a network of target genes. Silencing of all three factors in non-metastatic cells activated a program of metastasis-related genes, and increased metastasis in a transplantation model. Furthermore, the expression patterns of these factors strongly correlated with tumor progression in an autochthonous model of lung adenocarcinoma, and were closely associated with disease stage and survival outcomes of human patients. Collectively, these findings strongly argue that Nkx2-1, Foxa2, and Cdx2 synergize to restrain metastatic progression. Taken together, this study provides insights on some of the key molecular regulators of lung cancer metastasis. Our findings contribute to a better understanding of metastasis, and potentially to the development of better therapeutic strategies in the future.by Carman Man-Chung Li.Ph. D

Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

Although it has become increasingly clear that cancers display extensive cellular heterogeneity, the spatial growth dynamics of genetically distinct clones within developing solid tumours remain poorly understood. Here we leverage mosaic analysis with double markers (MADM) to trace subclonal populations retaining or lacking p53 within oncogenic Kras-initiated lung and pancreatic tumours. In both models, p53 constrains progression to advanced adenocarcinomas. Comparison of lineage-related p53 knockout and wild-type clones reveals a minor role of p53 in suppressing cell expansion in lung adenomas. In contrast, p53 loss promotes both the initiation and expansion of low-grade pancreatic intraepithelial neoplasia (PanINs), likely through differential expression of the p53 regulator p19ARF. Strikingly, lineage-related cells are often dispersed in lung adenomas and PanINs, contrasting with more contiguous growth of advanced subclones. Together, these results support cancer type-specific suppressive roles of p53 in early tumour progression and offer insights into clonal growth patterns during tumour development

Differential Tks5 isoform expression contributes to metastatic invasion of lung adenocarcinoma

Metastasis accounts for the vast majority of cancer-related deaths, yet the molecular mechanisms that drive metastatic spread remain poorly understood. Here we report that Tks5, which has been linked to the formation of proteolytic cellular protrusions known as invadopodia, undergoes an isoform switch during metastatic progression in a genetically engineered mouse model of lung adenocarcinoma. Nonmetastatic primary tumor-derived cells predominantly expressed a short isoform, Tks5short, while metastatic primary tumor- and metastasis-derived cells acquired increased expression of the full-length isoform Tks5long. This elevation of Tks5long to Tks5short ratio correlated with a commensurate increase in invadopodia activity in metastatic cells compared with nonmetastatic cells. Further characterization of these isoforms by knockdown and overexpression experiments demonstrated that Tks5long promoted invadopodia in vitro and increased metastasis in transplant models and an autochthonous model of lung adenocarcinoma. Conversely, Tks5short decreased invadopodia stability and proteolysis, acting as a natural dominant-negative inhibitor to Tks5long. Importantly, high Tks5long and low Tks5short expressions in human lung adenocarcinomas correlated with metastatic disease and predicted worse survival of early stage patients. These data indicate that tipping the Tks5 isoform balance to a high Tks5long to Tks5short ratio promotes invadopodia-mediated invasion and metastasis.National Institutes of Health (U.S.) (grant (5-U01-CA84306))National Cancer Institute (U.S.) (grant (P30-CA14051))Howard Hughes Medical Institute (Investigator)National Institutes of Health (U.S.) (grant R00-CA151968)National Institutes of Health (U.S.) (grant R01-CA175336)Virginia and Daniel K. Ludwig Graduate FellowshipMassachusetts Institute of Technology. Ludwig Center for Molecular Oncolog

Foxa2 and Cdx2 cooperate with Nkx2-1 to inhibit lung adenocarcinoma metastasis

Despite the fact that the majority of lung cancer deaths are due to metastasis, the molecular mechanisms driving metastatic progression are poorly understood. Here, we present evidence that loss of Foxa2 and Cdx2 synergizes with loss of Nkx2-1 to fully activate the metastatic program. These three lineage-specific transcription factors are consistently down-regulated in metastatic cells compared with nonmetastatic cells. Knockdown of these three factors acts synergistically and is sufficient to promote the metastatic potential of nonmetastatic cells to that of naturally arising metastatic cells in vivo. Furthermore, silencing of these three transcription factors is sufficient to account for a significant fraction of the gene expression differences between the nonmetastatic and metastatic states in lung adenocarcinoma, including up-regulated expression of the invadopodia component Tks5[subscript long], the embryonal proto-oncogene Hmga2, and the epithelial-to-mesenchymal mediator Snail. Finally, analyses of tumors from a genetically engineered mouse model and patients show that low expression of Nkx2-1, Foxa2, and Cdx2 strongly correlates with more advanced tumors and worse survival. Our findings reveal that a large part of the complex transcriptional network in metastasis can be controlled by a small number of regulatory nodes that function redundantly, and loss of multiple nodes is required to fully activate the metastatic program.National Cancer Institute (U.S.) (Cancer Center Support Grant P30-CA14051)Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Grant 5-U01-CA84306)United States. Dept. of Defense. Breast Cancer Research Program (U.S.) (Grant W81XWH-12-1-0031)Ludwig Center for Molecular Oncolog