PIAS1 and TIF1γ collaborate to promote SnoN SUMOylation and suppression of epithelial-mesenchymal transition.

SUMO E3 ligases specify protein substrates for SUMOylation. The SUMO E3 ligases PIAS1 and TIF1γ target the transcriptional regulator SnoN for SUMOylation leading to suppression of epithelial-mesenchymal transition (EMT). Whether and how TIF1γ and PIAS1 might coordinate SnoN SUMOylation and regulation of EMT remained unknown. Here, we reveal that SnoN associates simultaneously with both TIF1γ and PIAS1, leading to a trimeric protein complex. Hence, PIAS1 and TIF1γ collaborate to promote the SUMOylation of SnoN. Importantly, loss of function studies of PIAS1 and TIF1γ suggest that these E3 ligases act in an interdependent manner to suppress EMT of breast cell-derived tissue organoids. Collectively, our findings unveil a novel mechanism by which SUMO E3 ligases coordinate substrate SUMOylation with biological implications.

Defining and Targeting Adaptations to Oncogenic KRASG12C Inhibition Using Quantitative Temporal Proteomics.

Covalent inhibitors of the KRASG12C oncoprotein have recently been developed and are being evaluated in clinical trials. Resistance to targeted therapies is common and may limit long-term efficacy of KRAS inhibitors (KRASi). To identify pathways of adaptation to KRASi and predict drug combinations that circumvent resistance, we use mass-spectrometry-based quantitative temporal proteomics to profile the proteomic response to KRASi in pancreatic and lung cancer 2D and 3D cellular models. We quantify 10,805 proteins, representing the most comprehensive KRASi proteome (https://manciaslab.shinyapps.io/KRASi/). Our data reveal common mechanisms of acute and long-term response between KRASG12C-driven tumors. Based on these proteomic data, we identify potent combinations of KRASi with phosphatidylinositol 3-kinase (PI3K), HSP90, CDK4/6, and SHP2 inhibitors, in some instances converting a cytostatic response to KRASi monotherapy to a cytotoxic response to combination treatment. Overall, using quantitative temporal proteomics, we comprehensively characterize adaptations to KRASi and identify combinatorial regimens with potential therapeutic utility.

The PIAS3-Smurf2 sumoylation pathway suppresses breast cancer organoid invasiveness.

Tumor metastasis profoundly reduces the survival of breast cancer patients, but the mechanisms underlying breast cancer invasiveness and metastasis are incompletely understood. Here, we report that the E3 ubiquitin ligase Smurf2 acts in a sumoylation-dependent manner to suppress the invasive behavior of MDA-MB-231 human breast cancer cell-derived organoids. We also find that the SUMO E3 ligase PIAS3 inhibits the invasive growth of breast cancer cell-derived organoids. In mechanistic studies, PIAS3 maintains breast cancer organoids in a non-invasive state via sumoylation of Smurf2. Importantly, the E3 ubiquitin ligase activity is required for sumoylated Smurf2 to suppress the invasive growth of breast cancer-cell derived organoids. Collectively, our findings define a novel role for the PIAS3-Smurf2 sumoylation pathway in the suppression of breast cancer cell invasiveness. These findings lay the foundation for the development of novel biomarkers and targeted therapeutic approaches in breast cancer.

A novel role for the SUMO E3 ligase PIAS1 in cancer metastasis.

Tumor metastasis contributes to the grave morbidity and mortality of cancer, but the mechanisms underlying tumor cell invasiveness and metastasis remain incompletely understood. Here, we report that expression of the SUMO E3 ligase PIAS1 suppresses TGFß-induced activation of the matrix metalloproteinase MMP2 in human breast cancer cells. We also find that knockdown of endogenous PIAS1 or inhibition of its SUMO E3 ligase activity stimulates the ability of TGFß to induce an aggressive phenotype in three-dimensional breast cancer cell organoids. Importantly, inhibition of the SUMO E3-ligase activity of PIAS1 in breast cancer cells promotes metastases in mice in vivo. Collectively, our findings define a novel and critical role for the SUMO E3 ligase PIAS1 in the regulation of the invasive and metastatic potential of malignant breast cancer cells. These findings advance our understanding of cancer invasiveness and metastasis with potential implications for the development of biomarkers and therapies in breast cancer.

Identification of a Novel Link between the Protein Kinase NDR1 and TGFß Signaling in Epithelial Cells.

Transforming growth factor-beta (TGFß) is a secreted polypeptide that plays essential roles in cellular development and homeostasis. Although mechanisms of TGFß-induced responses have been characterized, our understanding of TGFß signaling remains incomplete. Here, we uncover a novel function for the protein kinase NDR1 (nuclear Dbf2-related 1) in TGFß responses. Using an immunopurification approach, we find that NDR1 associates with SnoN, a key component of TGFß signaling. Knockdown of NDR1 by RNA interference promotes the ability of TGFß to induce transcription and cell cycle arrest in NMuMG mammary epithelial cells. Conversely, expression of NDR1 represses TGFß-induced transcription and inhibits the ability of TGFß to induce cell cycle arrest in NMuMG cells. Mechanistically, we find that NDR1 acts in a kinase-dependent manner to suppress the ability of TGFß to induce the phosphorylation and consequent nuclear accumulation of Smad2, which is critical for TGFß-induced transcription and responses. Strikingly, we also find that TGFß reciprocally regulates NDR1, whereby TGFß triggers the degradation of NDR1 protein. Collectively, our findings define a novel and intimate link between the protein kinase NDR1 and TGFß signaling. NDR1 suppresses TGFß-induced transcription and cell cycle arrest, and counteracting NDR1's negative regulation, TGFß signaling induces the downregulation of NDR1 protein. These findings advance our understanding of TGFß signaling, with important implications in development and tumorigenesis.