Katsuta, Eriko

Background: Mechanically gated PIEZO channels lead to an influx of cations, activation of additional Ca2+ channels, and cell depolarization. This study aimed to investigate PIEZO2’s role in breast cancer. Methods: The clinical relevance of PIEZO2 expression in breast cancer patient was analyzed in a publicly available dataset. Utilizing PIEZO2 overexpressed breast cancer cells, and in vitro and in vivo experiments were conducted. Results: High expression of PIEZO2 was correlated with a worse survival in triple-negative breast cancer (TNBC) but not in other subtypes. Increased PEIZO2 channel function was confirmed in PIEZO2 overexpressed cells after mechanical stimulation. PIEZO2 overexpressed cells showed increased motility and invasive phenotypes as well as higher expression of SNAIL and Vimentin and lower expression of E-cadherin in TNBC cells. Correspondingly, high expression of PIEZO2 was correlated with the increased expression of epithelial–mesenchymal transition (EMT)-related genes in a TNBC patient. Activated Akt signaling was observed in PIEZO2 overexpressed TNBC cells. PIEZO2 overexpressed MDA-MB-231 cells formed a significantly higher number of lung metastases after orthotopic implantation. Conclusion: PIEZO2 activation led to enhanced SNAIL stabilization through Akt activation. It enhanced Vimentin and repressed E-cadherin transcription, resulting in increased metastatic potential and poor clinical outcomes in TNBC patients.

Abstract Background: PIM2 is overexpressed in hematopoietic and solid cancers, and is involved in multiple signaling pathways. In hematologic malignancies, PIM2 has been shown to be a pro-survival factor and demonstrated to be a possible therapeutic target. However, the role of PIM2 as a therapeutic target in solid cancers has not been fully elucidated. Methods: PIM2 expression was compared between normal and cancer tissues in The Cancer Genome Atlas (TCGA) datasets. Pan-PIM inhibitor treatment, as well as PIM2 overexpression/knockdown technique was utilized to investigate the PIM2 role in solid cancers. Multiple cancer models were utilized for in vitro and in vivo experiments to investigate if PIM2 as a therapeutic target and examine mechanism of anti-tumor activity by JP11646. Results: PIM2 was overexpressed in several types of cancer tissues, including breast, esophageal, head-neck, kidney, liver and endometrial cancers in TCGA. PIM2 overexpression promoted tumor growth in in vivo breast cancer mouse model. PIM2 inhibition by siRNA as well as pan-PIM inhibitor JP11646 activated apoptosis as seen by increased cleaved PARP. JP11646 treatment inhibited cancer cell proliferation in a dose-dependent manner in vitro in various types of solid cancers, including head-neck, ovarian, breast, prostate, liver, pancreatic, colorectal and non-small cell lung cancers. Interestingly, treatment with pan-PIM inhibitor JP11646 resulted in selective downregulation of PIM2 protein expression, but not PIM1 or PIM3 in breast cancer cell lines, BT549 and MDA-MB-231. The treatment also resulted in downregulation of phosphorylated downstream targets, 4EBP1 and TSC2 in breast cancer cells. However, additional proteasome inhibitor treatment blocked PIM2 downregulation and apoptosis activation was not seen. The result suggests that proteasome complex is required for PIM2 degradation which is necessary to induce apoptosis. JP11646 treatment inhibited tumor growth in breast, liver, pancreas, and non-small cell lung cancer in vivo xenograft models and was compared to multiple standard of care treatments showing advantage in lung tumors. Conclusion: We found that PIM2 promoted cancer progression in solid tumors. We show novel mechanism of JP11646 targeting PIM2 for degradation and induction of apoptosis. We show that PIM2 may serve as a novel therapeutic target for multiple types of solid cancers with enhanced therapeutic effect through protein degradation rather than kinase inhibition alone. Citation Format: Eriko Katsuta, Kazuaki Takabe, Alex Adjei, Mateusz Opyrchal. Novel mechanism of PIM2 inhibition leads to significant effect on solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6426.

Abstract Introduction: Breast cancer is the most common female malignancy. Despite improvements in therapeutic options, metastatic disease remains incurable with limited treatments especially in triple-negative breast cancer (TNBC). Clinical outcomes in patients with TNBC continue to be worse as compared to other breast cancer subtypes. Therefore, novel therapeutic targets for TNBC are in urgent need. PIEZO2, which is a transmembrane calcium channel, is known to function as a channel-type mechanosensor in various aspects of mechanotransduction. Recently there have been reports that PIEZO2 may be involved in carcinogenesis and cancer progression. However, the role of PIEZO2 in breast cancer has not been elucidated. We hypothesized that PIEZO2 plays significant roles in progression of TNBC. Methods: To investigate the role of PIEZO2 in human breast cancer patients, The Cancer Genome Atlas (TCGA) breast cancer cohort, in which there are mRNA expression data from RNA-seq and clinical data, was utilized. PIEZO2 expression level was compared among breast cancer subtypes. Patients’ survival was compared based on PIEZO2 expression in each subtype. We used gene set enrichment analysis (GSEA) algorithm to evaluate pathways associated with PIEZO2 expression. To further elucidate the mechanisms of PIEZO2 roles, TNBC cell line, MDA-MB-231 was utilized for in vitro experiments. PIEZO2 expression was downregulated by small interfering RNA (siRNA). Cell viability was quantified by MTT assay. Protein expression was compared by western blot. Results: Breast cancer showed the highest PIEZO2 expression level, among various types of cancers in TCGA. In the breast cancer cohort, PIEZO2 expression level was lower in the TNBC subtype compared to other subtypes (p<0.001). Thus we hypothesized that PIEZO2 has different roles in each subtype. Survival analyses revealed that patients with high expression of PIEZO2 tumors showed significantly worse overall survival (p=0.016) in the TNBC cohort. However, there was no significant difference in the hormone receptor (HR)-positive (p=0.555) and the HER2-positive (p=0.380) cohorts. To explore underlying mechanisms how PIEZO2 high expressing tumors associate with worse prognosis in TNBC, GSEA was conducted. PIEZO2 high expressing tumors enriched epithelial-mesenchymal transition (EMT; NES=1.90, p=0.012) and Angiogenesis (NES=1.91, p<0.001) gene sets, as well as TAZ/YAP gene set (NES=1.57, p=0.028). PIEZO2 knockdown by siRNA significantly suppressed cell viability. This finding was validated by PIEZO inhibitor GSMTx-4 treatment. Further, expression levels of EMT related proteins, such as Vimentin and Snail, were downregulated in the PIEZO2 knockdown MDA-MB-231 cells compared to control cells. Conclusion: We describe a novel signaling pathway mediated by PIEZO2 leading to worse clinical outcomes in patients with TNBC by activation of EMT. Further work is ongoing to validate PIEZO2 as a novel therapeutic target for TNBC. Citation Format: Eriko Katsuta, Marija Vujcic, Shipra Gandhi, Arthur Beyder, Kazuaki Takabe, Mateusz Opyrchal. Mechanosensing calcium channel PIEZO2 accelerates epithelial-mesenchymal transition in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-07-04.