通过靶向K-RAS通路的一个主要肿瘤脆弱性——Seven in absentia homolog (SIAH)——根除转移性癌症

Background: Persistent activation of the K-RAS pathway is multipotent: its downstream signaling networks promote tumorigenesis, drug resistance, relapse, and metastasis. Despite newly developed state-of-the-art therapies, none have yet to achieve durable antitumor efficacy. Seven in absentia homologues (SIAHs) are a conserved family of RING-domain E3 ubiquitin ligases that function as the most downstream signaling gatekeepers in the K-RAS pathway. Our preclinical studies demonstrated that SIAH inhibition leads to a tumor eradication phenotype of multiple stage IV human cancer cell lines both in vitro and in vivo. Aims: We propose SIAH is a major tumor vulnerability and actionable drug target for inhibiting K-RAS pathway activation. In this study, we aim to elucidate the molecular mechanisms underpinning the antitumor efficacy of our potent SIAH inhibitor, SIAH 2 PD , as a new targeted therapy to achieve tumor eradication. Methods: Reverse phase protein arrays (RPPAs) and principal component analysis (PCA) were used to quantify statistically significant fold-changes of 294 signaling proteins and phospho-proteins in response to SIAH blockade in five stage IV cell lines. RPPAs were performed in triplicate on MiaPaCa, MDA-MB-231, MDA-MB-468, HeLa, and A459 cell lines in which SIAH 2 PD expression was controlled by a doxycycline (DOX)-inducible Tet-ON/OFF system. Four experimental conditions were used: Tet-ON vector control cells without DOX ( group A ) and with DOX induction ( group B ); Tet-ON-SIAH 2 PD experimental cells without DOX ( group C ) and with DOX induction ( group D ). The ratios of each group were calculated in a pairwise comparison after normalization to GAPDH as an internal control. To validate putative targets of interest, immunofluorescence (IF), flow cytometry (FC), and Western blots (WB) were performed on cells for group C and D at 3-, 5-, and 7-days post-DOX-induction. Adherent and single cell suspensions were used respectively for cell-based assays in biological triplicates and normalized to alpha-Tubulin. Target protein expression was quantified and validated, and statistical analyses were performed by one-way ANOVA and T-tests using GraphPad Prism software. Results: The altered expression of NFκB, Caspase-3, Caspase-7, Cofilin, and PARP in response to SIAH blockade were independently validated by RPPA, WB, FC, and IF, demonstrating the roles of heightened cellular stress, apoptosis, and dysfunctional DNA damage and repair pathways induced by SIAH loss of function as mechanistic, synergistic to kill cancer cells and prevent tumor growth. Conclusions: RPPA cancer pathway mapping provides invaluable molecular insights into cancer network rewiring in response to SIAH blockade, revealing a major tumor vulnerability. This study supports the design of anti-SIAH-based, anti-EGFR/K-RAS targeted therapies to control and eradicate incurable human cancers.