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P1.02 - Biology/Pathology (ID 614)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.02-010 - Novel Role of hSSB2 in the Base Excision Repair Pathway (BER) (ID 9579)
09:30 - 09:30 | Presenting Author(s): Mark Adams
- Abstract
Background:
The base excision repair (BER) pathway is responsible for removing damaged or incorrectly incorporated uracil bases in the genome. Mismatched bases that persist in the genome and remain unrepaired may result in either lethal mutations or cytotoxic DNA double strand breaks. Previous studies have determined that hSSB1 is critical for the detection, signaling and repair of cytotoxic double strand DNA breaks and oxidized DNA lesions within the genome. The role of hSSB2 is, however, less clear. In this study, we have identified that the single stranded DNA binding proteins, hSSB1 and 2, are involved in the detection and removal of uracils within the genome and function as part of the BER pathway.
Method:
We identified a novel role for hSSB1 and hSSB2 in BER. EMSA and incision biochemical assays were used to determine the ability of hSSB1/2 to bind uracil containing mismatches. Incision assays were used to determine the effect hSSB2 and hSSB1 have on UNG2 activity. Two cytotoxic drugs (5-fluorouracil and pemetrexed), which induce uracil misincorporation in the genome, were used to determine the cell sensitivity in control and hSSB1/2-depleted cells using a live and dead cell assay. Immunoprecipitation, immunofluorescence and Protein-Protein interactions were carried out to determine whether hSSB2 and hSSB1 interacts with key regulatory proteins of the BER pathway.
Result:
This study demonstrates that hSSB1 and hSSB2 proteins can recognize and bind to double stranded DNA substrates containing a uracil mismatch. Interestingly, we have identified that hSSB1 and hSSB2 have a differential preference for uracil mismatches, with hSSB1 preferentially binding UA and hSSB2 UG mismatches. Furthermore, hSSB2 induces the incision activity of UNG2 by approximately two fold for a U:G mismatch but not a U:A mismatch. A549 lung adenocarcinoma cells depleted of both hSSB1 and hSSB2 are hypersensitive to 5-fluorouracil and pemetrexed. Loss of either hSSB1 or hSSB2 alone by siRNA results in a compensatory upregulation of hSSB2 or hSSB1 respectively, suggesting over-lapping functionality and substrate specificity.
Conclusion:
This study highlights the importance of hSSB2 and hSSB1 in the removal of uracil from the genome. Currently, pemetrexed and fluorouracil based agents are in use for treating lung cancer. This study raises the possibility that hSSB2 and hSSB1 may be biological indicators of response to fluorouracil and pemetrexed. Further, it may be possible to develop future hSSB2/hSSB1 inhibitors that could enhance the activity of these agents in the treatment of lung cancer.
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P1.02-071b - SASH1 Is a Prognostic Indicator and Future Target in NSCLC (ID 9591)
09:30 - 09:30 | Author(s): Mark Adams
- Abstract
Background:
Lung cancer is the most commonly diagnosed cancer in the world and the fifth most common in Australia, where it is responsible for almost one in five cancer deaths. SASH1 (SAM and SH3 domain-containing protein 1) is a tumor suppressor functioning to control of apoptosis and cellular proliferation. Previously SASH1 has been shown to be down-regulated in approximately 90% of lung cancers, however little is known about the role of SASH1 in the pathogenesis of the disease. Cytotoxic platinum based chemotherapy two-drug regimens remain a cornerstone NSCLC patient care, however, resistance to these agents is almost inevitable. The re-sensitisation of these cancer cells to chemotherapeutics is a key to improving patient survival. We hypothesised that modulation of SASH1 expression may alter cisplatin sensitivity.
Method:
A panel of lung cancer cell lines depleted of SASH1 (siRNA) or overexpressing SASH1 were analysed for protein levels via immunoblotting, cell proliferation, and survival/death assays. Treatment of lung cancer cells with the SASH1 protein stabilising compound chloropyramine (0-50 μM) and/or cisplatin (0-10 μM) was performed followed by immunoblotting for SASH1, cell proliferation, and survival/death assays. SASH1 IHC staining of adenocarcinoma and Squamous cell carcinomas was correlated with patient survival.
Result:
We demonstrated that SASH1 depletion results in a significant increase in cellular proliferation of NSCLC cancer cells. The depletion of SASH1 within lung cancer cell lines was associated with a significant increase in cisplatin resistance. Transfection of SASH1 into NSCLC cell lines induced cell death. The treatment of cells with the SASH1 protein stabilising compound chloropyramine increased SASH1 levels, reduced proliferation and induced apoptosis. Furthermore, chloropyramine increased cisplatin sensitivity. The relationship between SASH1 protein expression with overall survival was accessed in a NSCLC TMA panel. This showed that high SASH1 protein levels were associated with a poor prognosis in adenocarcinomas but were non-prognostic in squamous cell disease. Interestingly high SASH1 mRNA levels were associated with a favourable prognosis in adenocarcinoma but were not prognostic in squamous cell cancer. In a panel of cancer cell lines we observed no correlation between mRNA and protein levels that may explain this discrepancy.
Conclusion:
Agents that upregulate SASH1, or SASH1 gene therapy, are potential novel approaches to the management of NSCLC. Further preclinical and clinical studies of chloropyramine in combination with chemotherapy are justified in NSCLC.
