Employing a combination of quantitative proteomics technology and traditional molecular cell biology approaches, our laboratory studies 1) the secreted mediators of cellular senescence, 2) the ubiquitination substrates and interaction partners of a tumor-suppressing ubiquitin ligase, VHL, and 3) the biomarkers for VHL-mutated and BRCA1-mutated cancer.
1) Cellular senescence plays key roles in tumor suppression and organismal aging. Accumulating evidence suggests profoundly altered protein secretion from senescent cells, which may recruit immune cells for clearance of senescent cells, affect the architecture or function of surrounding tissues, modulate tumor progression, and contribute to aging and age-related diseases. Using quantitative proteomic analysis of protein secretion from senescent cells, we identified two secreted mediators of senescence, SFRP1 and IGFBP3. (NIH R21AG029587)
We found that fibroblasts induced to senesce by DNA damage over-secrete SFRP1 (Secreted Frizzled-related Protein 1), a secreted antagonist of Wnt signaling and tumor suppressor. SFRP1 mediated senescence phenotypes through inhibition of Wnt signaling and activation of the Rb pathway. The role of Wnt inhibition in cellular senescence was also supported by senescence induction by different Wnt antagonists (SFRP1-5 and DKK1), by pharmacological inhibition of Wnt signaling, and by knock-down of β-catenin. Interestingly, cancer-associated SFRP1 mutants were defective for senescence induction. These results suggest that SFRP1 is an extracellular component of stress-induced senescence signaling that responds to potentially carcinogenic stresses such as DNA damage and induces cellular senescence in an autocrine and paracrine fashion, which may lead to non-cell autonomous tumor suppression.
In addition to normal cells, cancer cells also undergo senescence upon chemotherapeutic drug treatment. We found that MCF-7 breast cancer cells induced to senesce by doxorubicin treatment display elevated extracellular IGFBP3 (Insulin-like Growth Factor Binding Protein 3), a secreted inhibitor of IGF signaling. We determined that IGFBP3 induces senescence through suppression of Akt kinase signaling and requiring the Rb and p53 pathways. To dissect the biochemical pathways regulating IGFBP3, we undertook a proteomic screen for IGFBP3-interacting proteins and identified t-PA (tissue-type plasminogen activator) as interactor. t-PA is a protease that cleaves plasminogen. We found that t-PA can also cleave IGFBP3 and counteract the senescence induction by IGFBP3. The protease activity of t-PA is specifically inhibited by PAI-1 (plasminogen activator inhibitor 1). We found that PAI-1 also inhibits IGFBP3 cleavage by t-PA and induces senescence. PAI-1 was previously identified as a mediator of cellular senescence and by using shRNA-mediated knockdown of IGFBP3, we demonstrated that IGFBP3 is a critical downstream target of PAI-1-induced senescence. These results suggest a role for extracellular PAI-1 – t-PA – IGFBP3 cascade in the regulation of stress-induced senescence.
Interestingly, IGFBP3 is specifically silenced in Ewing sarcoma, a childhood cancer of bone and soft tissues, and its re-expression potently inhibits Ewing sarcoma growth. We have identified the downstream targets of IGFBP3 in Ewing sarcoma cells and are characterizing their roles in mediating anti-Ewing sarcoma effect.
2) Mutation of the VHL tumor suppressor plays a central role in the generation of both hereditary and non-hereditary kidney cancers. VHL is a ubiquitin ligase, an enzyme that attaches a small protein called ubiquitin to its substrate proteins. A major bottleneck in understanding the functions of ubiquitin ligases has been the difficulty in identifying their ubiquitination substrates. We are using quantitative proteomics approaches to identify the ubiquitination substrates and interaction partners for VHL. (NIH R01CA125020)
3) The serum cancer biomarkers that can be measured by a simple blood test have great potential for early diagnosis, disease monitoring, and assessment of therapeutic response. However, direct proteomic analysis of cancer patient serum is technically difficult. As an alternative, we are identifying candidate cancer biomarkers by analyzing proteins secreted from cancer cells in culture, which will be validated using serum samples from cancer patients and healthy controls. (NIH R21CA139170)