Abstract
Introduction
Pancreatic ductal adenocarcinoma (PDAC) and triple-negative breast cancer (TNBC) remain the deadliest forms of human cancer and represent a major unmet clinical need, particularly in the residual, relapsed, multidrug-resistant, and metastatic settings, where late-stage PDAC and TNBC are still undruggable and incurable, despite new breakthrough therapies. Targeting the most downstream gatekeeper of the oncogenic EGFR/K-RAS pathway, Seven in Absentia Homologue (SIAH), represents an innovative therapeutic strategy. Expression of a dominant-negative SIAH inhibitor of SIAHWT, hSIAH2PD, induces cell death, cellular stress, increased DNA damage response (DDR), and abolishes tumor growth in mice, i.e. xenograft and patient-derived xenograft (PDX) models of human cancer. Encouraged by these published and preliminary results, we aim to delineate the molecular mechanisms underlying tumor eradication induced by SIAH inhibition. We used reverse-phase protein array (RPPA) analysis to conduct global cancer pathway mapping and to identify statistically significant increases in DDR and apoptotic phospho-proteins/proteins. These results were validated by Immunofluorescence (IF) staining and/or Western blotting. A three-dimensional (3D) tumor growth assay in soft agar was performed to quantify the effects of SIAH inhibition on tumor growth and destruction longitudinally.
Methods
RPPA analysis was performed in biological triplicate to identify molecular alterations in SIAH-proficient and SIAH-deficient MiaPaCa-2 and MDA-MB-231 cells. IF staining and Western blotting were performed at 3- and 7-days following DOX induction to validate statistically significant increases in several DDR- and apoptosis-associated proteins induced by SIAH inhibition. MiaPaCa-2 tumors in soft agar were grown utilizing a microplate format and subjected to longitudinal tumor tracking for 24-days utilizing the Cytation C10. ImageJ was utilized to quantify tumor volume and generate tumor growth curves. Matched brightfield and Z-stack imaging were utilized to visualize tumor morphology and GFP-tagged SIAH inhibitor expression.
Results
RPPA analysis identified marked changes in DDR and apoptosis following SIAH inhibition. In MiaPaCa-2 cells, RPPA demonstrated statistically significant increases in p53S15, cleaved PARP, and cleaved caspases-3/7, which were successfully validated by IF and/or Western blotting. In MDA-MB-231 cells, RPPA analysis showed statistically significant increases in cleaved PARP and cleaved caspase-7, which were successfully validated by IF and/or Western blotting. Furthermore, longitudinal tumor tracking of MiaPaCa-2 tumoroids showed a clear tumor destruction phenotype following SIAH inhibition. Quantitative ImageJ analysis demonstrated a significant reduction in 3D tumor volume induced by SIAH inhibition (p0.0008). Normalization to baseline tumor volume to account for inherent variation in initial 3D tumoroids yielded an even more statistically significant result (p0.000000002).
Conclusion
Here, we report that SIAH inhibition activated DNA damage-associated apoptotic pathways in human cancer cell lines. Furthermore, quantitative longitudinal 3D analysis demonstrated gradual tumor destruction following SIAH inhibition in MiaPaCa-2 cells, highlighting the efficacy of the SIAH inhibitor as a new targeted therapy to eradicate oncogenic EGFR/RAS-driven cancers. Ultimately, delineating the relationship between SIAH inhibition, increased DDR, apoptotic responses, and 3D tumor destruction will further support our central hypothesis that SIAH represents a promising new therapeutic target for the treatment of “undruggable and incurable” human malignancies.