Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease that can be separated into distinct subtypes based on molecular signatures. Identifying PDAC subtype-specific therapeutic vulnerabilities is necessary to develop precision medicine approaches to treat PDAC. Experimental Design: A total of 56 PDAC liver metastases were obtained from the UNMC Rapid Autopsy Program and analyzed with quantitative proteomics. PDAC subtypes were identified by principal component analysis based on protein expression profiling. Proteomic subtypes were further characterized by the associated clinical information, including but not limited to survival analysis, drug treatment response, and smoking and drinking status. Results: Over 3,960 proteins were identified and used to delineate four distinct PDAC microenvironment subtypes: (i) metabolic; (ii) progenitor-like; (iii) proliferative; and (iv) inflammatory. PDAC risk factors of alcohol and tobacco consumption correlate with subtype classifications. Enhanced survival is observed in FOLFIRINOX treated metabolic and progenitor-like subtypes compared with the proliferative and inflammatory subtypes. In addition, TYMP, PDCD6IP, ERAP1, and STMN showed significant association with patient survival in a subtype-specific manner. Gemcitabine-induced alterations in the proteome identify proteins, such as serine hydroxymethyltransferase 1, associated with drug resistance. Conclusions: These data demonstrate that proteomic analysis of clinical PDAC liver metastases can identify molecular signatures unique to disease subtypes and point to opportunities for therapeutic development to improve the treatment of PDAC.

BRCA1 C-terminal domains are found in a specialized group of 23 proteins that function in the DNA damage response to protect genomic integrity. C-terminal domain phosphatase 1 (CTDP1) is the only phosphatase with a BRCA1 C-terminal domain in the human proteome, yet direct participation in the DNA damage response has not been reported. Examination of the CTDP1 BRCA1 C-terminal domain-specific protein interaction network revealed 103 high confidence interactions enriched in DNA damage response proteins, including FANCA and FANCI that are central to the Fanconi anemia DNA repair pathway necessary for the resolution of DNA interstrand crosslink damage. CTDP1 expression promotes DNA damage-induced FANCA and FANCD2 foci formation and enhances homologous recombination repair efficiency. CTDP1 was found to regulate multiple aspects of FANCI activity, including chromatin localization, interaction with γ-H2AX, and SQ motif phosphorylations. Knockdown of CTDP1 increases MCF-10A sensitivity to DNA interstrand crosslinks and double-strand breaks, but not ultraviolet radiation. In addition, CTDP1 knockdown impairs in vitro and in vivo growth of breast cancer cell lines. These results elucidate the molecular functions of CTDP1 in Fanconi anemia interstrand crosslink repair and identify this protein as a potential target for breast cancer therapy.

Deleterious mutations in Breast Cancer 1 (BRCA1) are associated with an increased risk of breast and ovarian cancer. Mutations in the tandem BRCA1 C-terminal (tBRCT) protein domain disrupt critical protein interactions required for the faithful repair of DNA through homologous recombination, which contributes to oncogenesis. Our studies have identified RICTOR, PRR5, and SIN1 subunits of the mammalian target of rapamycin complex 2 (mTORC2) as interacting partners with the tBRCT domain of BRCA1 leading to the disruption of the mTORC2 complex. However, the interplay between mTORC2 signaling and BRCA1 function in the DNA damage response (DDR) remains to be determined. In this study, we used protein interaction assays to determine the binary interactions between the tBRCT domain and mTORC2 subunits, evaluated the impact of mTOR inhibition on the transcriptional function of the tBRCT, evaluated the impact of mTOR signaling on BRCA1 recruitment to DNA damage-induced foci and determined the breast cancer cell line response to mTOR inhibition dependent upon BRCA1 expression and mutation. This study determined that PRR5, RICTOR, and SIN1 could each independently interact with the BRCA1 tBRCT. Inhibition of mTORC1, but not mTORC1/2, increases BRCA1 transcriptional activation activity. Treatment with pan-mTOR inhibitor PP242 diminishes DNA damage-induced γH2AX and BRCA1 foci formation. Breast cancer cells lacking expression of functional BRCA1 are more sensitive to mTOR inhibitors. These data suggest that mTOR signaling is required for BRCA1 response to DNA damage and breast cancer cells lacking BRCA1 are more sensitive to pan-mTOR inhibition. This work suggests chemotherapeutic strategies using mTOR inhibitors could be tailored for patients that lack functional BRCA1.

Hyperinsulinemia affects 72% of Fanconi anemia (FA) patients and an additional 25% experience lowered glucose tolerance or frank diabetes. The underlying molecular mechanisms contributing to the dysfunction of FA pancreas β cells is unknown. Therefore, we sought to evaluate the functional role of FANCA, the most commonly mutated gene in FA, in glucose-stimulated insulin secretion (GSIS). This study reveals that FANCA or FANCB knockdown impairs GSIS in human pancreas β cell line EndoC-βH3. To identify potential pathways by which FANCA might regulate GSIS, we employed a proteomics approach to identify FANCA protein interactions in EndoC-βH3 differentially regulated in response to elevated glucose levels. Glucose-dependent changes in the FANCA interaction network were observed, including increased association with other FA family proteins, suggesting an activation of the DNA damage response in response to elevated glucose levels. Reactive oxygen species increase in response to glucose stimulation and are necessary for GSIS in EndoC-βH3 cells. Glucose-induced activation of the DNA damage response was also observed as an increase in the DNA damage foci marker γ-H2AX and dependent upon the presence of reactive oxygen species. These results illuminate the role of FANCA in GSIS and its protein interactions regulated by glucose stimulation that may explain the prevalence of β cell-specific endocrinopathies in FA patients.

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