Development of surface proteome signature technology and identification of CD44 as an anti-apoptotic mediator

Systemic treatment of cancer with cytotoxic agents is a crucial component of therapeutic oncology. Acquired or inherent cellular resistance mechanisms to these compounds greatly reduce their efficacy and are associated with poor prognosis. Identification of biomarkers that can predict tumor response to chemotherapy would allow clinicians to better tailor treatment regimens for optimization of their cytotoxic effects.;The first part of this thesis describes the development of a novel surface proteome profiling technology, Surface Proteome Signatures (SPS), and its use in identifying biomarkers for drug resistance. SPS utilizes a diverse single-chain, variable fragment (scFv) antibody library to generate quantifiable fluorescent signals that correlate directly to antigen expression on the cell surface. We generated a 70-scFv surface proteome signature for two cell lines differing in their response to doxorubicin and identified CD44 as being overexpressed on the cell membrane of resistant cells. Subsequent experiments confirmed a role for CD44 in drug resistance through upregulation of the anti-apoptotic protein Bcl-xL in response to stimulus by its ligand hyaluronan.;The second part of this work expands upon CD44's anti-apoptotic function by examining specific isoforms implicated in apoptotic resistance. While CD44 standard has been shown to confer resistance to both extrinsic and chemotherapeutic apoptosis, little is known about what role its isoforms play in this process. We chose to explore this aspect of CD44 function in apoptosis resistant Breast Cancer Initiating Cells (BCIC) by examining the function of CD44 isoforms V5, V6 and V9. QRT-PCR analysis on SUM159 BCIC showed that these isoforms are differentially expressed in BCIC compared to the parental cell population. The subsequent depletion of these isoforms enhanced BCIC susceptibility to FAS mediated apoptosis, but not to treatment with the chemotherapeutic drug doxorubicin.;The work presented here describes a novel technology capable of generating large-scale surface protein expression profiles for use in biomarker identification. In our application of SPS, we identified CD44 as a biomarker for drug resistance and confirmed its hyaluronan dependent upregulation of the anti-apoptotic protein Bcl-xL. Our studies in SUM 159 BCIC demonstrated, for the first time, differential expression of CD44 isoforms in BCIC and showed they have a functional role in resistance to FAS mediated apoptosis in these cells.