Differences in the mutations underiying an individual's cancer can dramatically affect the best treatment choice''and it is becoming clear that different ethnic populations differ significantly in which mutations drive their lung cancers^'^. Lung cancer is the leading cancer killer among Puerto Rican (PR) men and second killer among PR women*'^. Despite this fact, little is known regarding the molecular mechanisms driving lung cancer among PRs. For example, recent work has shown that the rate of epidermal growth factor receptor {EGFF^ mutations in Latin American populations is significantiy higher than V /hites and African Americans^. This information is important as EGFR mutations are targetable in the clinic^'^?^. This application proposes to establish a lung cancer molecular database on ~100 PR lung cancer patients. The work described herein will specifically address the hypothesis that PR lung cancer patients have a different pattern of mutations in genes that are most commonly mutated in the White, mainland US population. Experiments will assess alterations in KRAS, TP53, EGFR, BFiAF, CDKN1C and RBI. if this hypothesis is verified, it could dramatically affect genetic testing and treatment recommendations for PR lung cancer patients since many of these mutations can be targeted clinically?. Perhaps most importantiy, these efforts will create a significant data and tissue repository that will benefit future research on the leading cancer killer of PR men. This proposal will also probe the role of the retinoblastoma {RBI) pathway in lung cancer. RBI was the first tumor suppressor gene to be discovered, and yet its potency as a tumor suppressor remains only partially explained. In exciting new experiments, we have uncovered a role for pRb (the protein product of the RB1 gene) in the regulation of cell-to-cell interactions?. This is a novel role since pRb is predominantiy known as a cell-cycle regulator. pRb is found to be required for the regulated expression of cadherins, which are components of the adherens junction structures involved in cellular adhesion. Abnormal cadherin expression due to pRb loss resulted in cells with disrupted adherens junctions and impaired adhesive properties. Expression microarrays comparing Rb+/+ and Rb-/- cells show that pRb impacts the transcription of a wide repertoire of cell adhesion-related genes, including various integrins and cadherins^ Importantly, the examination of publically available gene expression datasets demonstrates that the expression levels of a subset of these pRb-regulated cell adhesion genes strongly correlates with overall survival in lung adenocarcinoma (AC). This suggests that aberrant cell adhesion-related gene expression, possibly due to pRb inactivation (directiy or as a result of CDKN2A silencing), could be related to the molecular etiology of AC. This application has three specific aims.
The first aim will focus on creating a molecular database corresponding to -100 PR NSCLC (non-small-cell lung cancer) patients using tumor-derived DNA. First, we will identify the mutations present in genes commonly mutated genes in NSCLC (including KFiAS, TP53, EFGR, RB1, B-RAF and ALK fusions;. In addition, we will monitor deregulation of the RBI pathway by measuring CDKN2A promoter methylation and CDKN2A gene rearrangements.
The second aim will utilize tumor-derived mRNA from the same -100 patients for microarray-based gene expression analysis. We will use clustering approaches to draw correlations between the mutation patterns (observed in Aim 1) with expression profileis (observed in Aim 2). We hypothesize that genetic alterations of the CDKN2A/RB1 pathway may be found to correlate well with the deregulation of Rb-regulated celi adhesion genes. Finally, the third aim will focus on the basic biology of how pRb affects cell-to-cell adhesion at the cellular and molecular levels. Specifically, this aim is focused on the characterization of the molecular mechanisms by which pRb promotes cell adhesion from its nuclear position. The hypothesis that we will test in Aim 3 is that pRb impinges on cell adhesion by regulating the assembly and stabilization of adherens junctions at the cell membrane in a manner that involves the small Rho GTPase Rac 1.
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