BackgroundThe response of soft tissue sarcomas to cytotoxic chemotherapy is inconsistent. Biomarkers of chemoresistance or chemosensitivity are needed in order to identify appropriate patients for treatment. Given that many chemotherapeutics kill cells through direct DNA interactions, we hypothesized that upregulation of DNA damage response mechanisms would confer resistance to cytotoxic chemotherapy in sarcomas. To study this, we used spontaneously-occurring feline injection-site sarcomas (FISS).MethodsγH2AX and p53 expression were determined in biopsy samples of FISS. γH2AX expression was determined via immunohistochemistry whereas p53 expression was determined via qRT-PCR. Cell lines derived from these sarcoma biopsies were then treated with carboplatin (N = 11) or doxorubicin (N = 5) and allowed to grow as colonies. Colony forming-ability of cells exposed to chemotherapy was compared to matched, untreated cells and expressed as percent survival relative to controls. ImageJ was used for quantification. A mixed model analysis was performed to determine if an association existed between relative survival of the treated cells and γH2AX or p53 expression in the original tumors. Cell lines were validated via vimentin expression or growth as subcutaneous sarcomas in nude mice.ResultsAn association was detected between γH2AX expression and relative survival in cells exposed to carboplatin (P = 0.0250). In the 11 FISS tumors evaluated, γH2AX expression ranged from 2.2 to 18.8% (mean, 13.3%). Cells from tumors with γH2AX expression higher than the sample population mean had fourfold greater relative survival after carboplatin exposure than cells from tumors with γH2AX expression less than the mean. There was no association between relative survival after carboplatin exposure and p53 expression (P = 0.1608), and there was no association between relative survival after doxorubicin exposure and either γH2AX (P = 0.6124) or p53 (P = 0.8645) expression. Four cell lines were validated via growth as sarcomas in nude mice. Vimentin expression was confirmed in the other 7 cell lines.ConclusionsγH2AX expression, but not wild type p53, may potentially serve as a biomarker of resistance to platinum therapeutics in soft tissue sarcomas. To further investigate this finding, prospective, in vivo studies are indicated in animal models.

The DNA damage response (DDR) factor HUS1 is vital for proper functioning of the ATR checkpoint pathway. Following DNA damage, HUS1 forms a heterotrimeric complex with RAD9 and RAD1 and works with other checkpoint and scaffold proteins to activate ATR and CHK1 kinases. HUS1 also has separate functions that promote DNA repair. Due to its essential nature and diverse roles in genome maintenance, we hypothesized that HUS1 would be critical in the response of normal and neoplastic tissues to the DNA-damaging chemotherapeutic, cisplatin. Cisplatin is a widely used chemotherapeutic that damages DNA via platinated adducts, which form intrastrand and interstrand crosslinks. To test our hypothesis, we made use of a mouse model whereby a hypomorphic Hus1 allele is combined with a null allele in order to achieve partial impairment (Hus1neo/Δ1n) in vivo. Hus1+ and Hus1neo/Δ1n FVB mice were administered high dose cisplatin intraperitoneally (13.5 mg/kg). Body weights were recorded daily and mice were euthanized when humane intervention criteria were met. Hus1neo/Δ1n mice had reduced 7-day survival (P = .0071) and increased weight loss (P = .0485) when compared to Hus1+ mice. In order to determine if neoplastic tissues had a similar requirement for HUS1 in response to DNA damage inflicted by cisplatin, we combined Hus1 impairment with K-ras oncogene activation in vivo. K-RAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC). Mutations in this oncogene, the majority of which occur in codon 12, affect 25–30% of western NSCLC patients. K-RAS encodes for a membrane-bound GTPase that regulates cell growth, differentiation, and survival. In our model, activation of oncogenic K-ras is restricted to the lung and is achieved via a tetracycline-controlled reverse transactivator linked to the CCSP promoter. In the presence of doxycycline, mutant Kras4bG12D is expressed in type II alveolar epithelial cells, and lung tumors develop in a regulatable, predictable manner. Mice with wild-type or partial Hus1 impairment and simultaneous oncogenic K-ras activation were screened for lung tumor development via computed tomography (micro-CT), then treated with maximum-tolerated dose (MTD) cisplatin, and assessed for response according to semiautomated volumetric methods and the clinical standard, RECIST (Response Evaluation Criteria in Solid Tumors). Using either histologic evaluation or micro-CT imaging, mice with partial Hus1 impairment have a decreased tumor burden at 1- and 3- weeks after cisplatin treatment, as compared to mice with wild type Hus1. Overall, this data identifies a role for HUS1 in the response of normal and neoplastic tissues to the DNA-damaging chemotherapeutic, cisplatin. Understanding tissue specific responses to cisplatin is important for determining how therapeutics targeting the DDR will work cooperatively with traditional cytotoxic agents in the treatment of cancer. This abstract is also being presented as Poster B08. Citation Format: Terese E. Noe, Cleo C. Siderides, Aitor Gallastegui, James M. Cheung, Tina Abratte, Kelly R. Hume. Normal and neoplastic tissues with partial Hus1 impairment show hypersensitivity to cisplatin in vivo [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR16.