Editorial on the Research TopicSirtuins from Bench to Bedside: How Far Are We?Sirtuins (SIRT1-7) regulate diverse physiological processes including metabolism, oxidative stress, DNA repair, protein synthesis, inflammation, and cell death. Recent literature shows that dysregulation of sirtuins could lead to heart failure, neurological disorders, and cancer. However, the mechanisms of sirtuin regulation in diverse cellular processes and their clinical utility are not well understood; five reviews and a research report examining these topics are discussed here.Watroba and Szukiewicz focused on the involvement of sirtuins in neurodegeneration, the inflammatory response, metabolic syndrome, DNA damage, and genomic instability as hallmarks of aging and cancer incidence. It explains that caloric restriction could play a role in anti-aging by activating sirtuins, which stimulate nicotinamide adenine dinucleotide (NAD+) biosynthesis, raising the level of intracellular NAD+. This is a hot topic due to public and scientific interest in caloric restriction to reduce body weight and slow aging and associated diseases. SIRT1 induction in neurons results in mTOR inhibition that promotes neurite outgrowth and increased degradation of the toxic protein aggregates like beta-amyloid that are responsible for neurodegeneration. As there are currently no drugs available to prevent neurodegeneration, sirtuin isoforms should be further explored for neuroprotection. Authors discussed some tissue-specific functions of sirtuins like antiapoptotic, anti-inflammatory, and anti-fibrotic actions in the kidneys, liver, heart, skeletal muscle, hematopoietic system, and immune system.The genetic and pharmacological modulation of sirtuins in cancer, neurological, and cardiovascular diseases (CVD) was the focus of Hong and Lin. The reviewers highlighted modulators strategically designed to directly bind sirtuins and generate beneficial effects in cellular and animal models of human disease. Sirtuins are involved in a plethora of biological pathways and could play both tumor suppressor and activator roles. SIRT1, SIRT2, SIRT3, and SIRT5 inhibitors were developed to prevent cell proliferation and tumor growth, and numerous pan-sirtuin inhibitors also decrease cancer cell proliferation. Many SIRT2 inhibitors ameliorate the symptoms from neurological disease. In addition, several sirtuins have been connected to CVDs, including vascular aging, atherosclerosis, and cardiac hypertrophy. Although the authors noted a few contradicting reports, some general trends can be extracted from the literature. SIRT2 and SIRT5 inhibitors show consistent and promising effects in treating cancers and SIRT2 inhibitors show beneficial effects in neurological diseases. Conversely, SIRT1 and SIRT6 inhibitors aggravate CVDs, showing the need for reliable SIRT1 and SIRT6 activators. These trends support the development of sirtuin modulators with enhanced potency and selectivity, to validate preclinical data and potentially treat various human diseases.Wang et al. summarized t...

Background: Metaplastic breast cancer (MpBC) is a rare, lethal, and highly chemoresistant breast cancer subtype, with no FDA-approved therapeutic options. Most MpBCs are triple-negative, yet have a worse prognosis than non-metaplastic triple-negative breast cancer (non-MpTNBC). MpBC tumors are enriched for markers of epithelial-to-mesenchymal transition (EMT)/cancer stem cells (CSC), produce high nitric oxide (NO) levels, and have a hyperactive phosphoinositide 3-kinase (PI3K) signaling pathway. Increased PI3K and inducible nitric oxide synthase (iNOS) activity are poor prognostic indicators in MpBC. NO can activate multiple oncogenic pathways spatially and temporally, such as PI3K and transforming growth factor beta (TGFβ), a critical regulator of EMT. Therefore, our study evaluates whether pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) augments the efficacy of alpha isoform-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Methods: MpBC cell lines (SUM159, BT549, Hs578T, HCC1806) and Patient-Derived Xenograft (PDX) models were used in our studies. Droplet digital polymerase chain reaction (ddPCR) was conducted to evaluate the iNOS-associated mutation (RPL39 A14V) and PIK3CA hotspot mutation rates in PDX models. Cell viability (SRB/Cell Titer-Glo), combination index (CI), immunoblotting, and immunofluorescence of treated MpBC cell lines and tumor tissues were evaluated. Results: Immunostaining analysis revealed that MpBC PDX tumors had elevated co-expression of iNOS and pAkt (60% vs 23%, p=0.0495) relative to non-MpTNBC PDX tumors. MpBC PDX tumors had higher RPL39 A14V (66% vs 4.7%, p< 0.0006) and PIK3CA hotspot mutation rates (50% vs 19.1%, p=0.1247) than non-MpTNBC PDX tumors. Combining L-NMMA with alpelisib was synergistic in MpBC cell lines harboring PIK3CA/PIK3R1 mutations (CI< 1) and antagonistic in PIK3CA-wild type and PTEN-deleted models (CI>1). In vivo evaluation using MpBC PDX tumors found that L-NMMA significantly augmented the efficacy of alpelisib in reducing tumor volume in PIK3CA-mutated MpBC PDX models. Transcriptomic analysis found gene sets associated with EMT reversal, such as the formation of cornified envelope (Padj = 0.0254) and keratinization pathway (Padj = 0.048) were enriched pathways in MpBC PDX tumors that responded to combination therapy. Pharmacological and genomic inhibition of iNOS reversed EMT in MpBC cells, as shown by decreased expression of Zeb1, TGFβ, Snail, Vimentin, and increased expression of E-cadherin and ZO-1 in immunoblotting analysis. MpBC cells with NOS2 knockout acquired an epithelial-like cellular morphology and this reversal of EMT rendered MpBC cells more sensitive to alpelisib and taxane-chemotherapy. MpBC PDX tumors that responded to combination therapy also exhibited a reversal in EMT, with an associated decrease in aldehyde dehydrogenase (ALDH1), a CSC marker. L-NMMA and alpelisib therapy also resulted in the loss of tumor-initiating ability, enhanced chemosensitivity, and improved overall survival in MpBC PDX models. These studies paralleled results from a phase 1b/2 clinical trial with L-NMMA combined with taxane chemotherapy in a cohort of anthracycline-refractory MpBC patients (n=15, NCT02834403). The clinical benefit rate was 40% (6/15), the overall response rate was 20% (3/15), and one patient achieved a pathologic complete response. Relative to baseline tumors, the responder end-of-treatment tumors had undergone reversal of EMT, with enhanced expression of E-cadherin, and decreased expression of iNOS, Zeb1, and ALDH1. Conclusion: Our findings suggest that combining L-NMMA and alpelisib is a novel therapeutic strategy to treat MpBC by reversing EMT and decreasing CSCs, rendering MpBC tumors more chemosensitive. This combination therapy is being tested in a first multicenter phase 2 study targeting this orphan disease. Citation Format: Tejaswini Reddy, Akshjot Puri, Liliana Guzman, Wei Qian, Jianying Zhou, Roberto Rosato, Hong Zhao, Christoforos Thomas, Xiaoxian Li, Bijan Mahboubi, Adrian Oo, Young-Jae Cho, Baek Kim, Jose Thaiparambil, Camila Ayerbe, Noah Giese, Stacy Moulder, Helen Piwnica-Worms, Funda Meric-Bernstam, Jenny Chang. NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-05.

Background Unlike normal cells, cancer cells frequently have multiple centrosomes that can cluster to form bipolar mitotic spindles and allow for successful cell division. Inhibiting centrosome clustering, therefore, holds therapeutic promise to promote cancer cell‐specific cell death. Methods We used confocal microscopy, real‐time PCR, siRNA knockdown, and western blot to analyze centrosome clustering and declustering using normal lung bronchial epithelial and nonsmall‐cell lung cancer (NSCLC) cell lines. Also, we used Ingenuity Pathway Analysis software to identify novel pathways associated with centrosome clustering. Results In this study, we found that exposure to cigarette smoke condensate induces centrosome amplification and clustering in human lung epithelial cells. We observed a similar increase in centrosome amplification and clustering in unexposed NSCLC cell lines which may suggest a common underlying mechanism for lung carcinogenesis. We identified a cyclin D2‐mediated centrosome clustering pathway that involves a sonic hedgehog‐forkhead box protein M1 axis which is critical for mitosis. We also observed that cyclin D2 knockdown induced multipolar mitotic spindles that could eventually lead to cell death. Conclusions Here we report a novel role of cyclin D2 in the regulation of centrosome clustering, which could allow the identification of tumors sensitive to cyclin D2 inhibitors. Our data reveal a pathway that can be targeted to inhibit centrosome clustering by interfering with the expression of cyclin D2‐associated genes.

Metaplastic breast cancer (MpBC) is a rare and highly chemoresistant breast cancer subtype, with a median survival of 8 months after metastatic disease, and no standard therapeutic options. MpBCs are enriched for epithelial-to-mesenchymal transition (EMT)/cancer stem cell (CSC) markers, produce high levels of nitric oxide (NO), and have a hyperactive phosphoinositide 3-kinase (PI3K) signaling pathway. NO activates multiple oncogenic pathways, such as PI3K and transforming growth factor beta (TGFβ), a regulator of EMT. Therefore, we hypothesized that pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) could augment the efficacy of α-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Immunostaining analysis revealed that MpBC PDX tumors had elevated co-expression of iNOS and pAkt (60% vs 23%, p=0.04) relative to triple-negative breast cancer (TNBC) PDX tumors. MpBC PDX tumors had higher RPL39 A14V (66% vs 4.7%, p< 0.00) and PIK3CA hotspot mutation rates (50% vs 19.1%, p=0.31) than TNBC PDX tumors. L-NMMA was synergistic with alpelisib in MpBC cell lines with PIK3CA/PIK3R1 mutations and antagonistic in PIK3CA-wild type and PTEN-deleted models. In vivo evaluation using MpBC PDX tumors found that L-NMMA augmented the efficacy of alpelisib in reducing tumor volume in PIK3CA-mutated MpBC PDX models. Transcriptomic analysis found gene sets associated with EMT reversal, such as the formation of cornified envelope (NES = 2.04 Nom p<0.00) and keratinization pathway (NES = 2.06, Nom p<0.00), were enriched pathways in MpBC PDX tumors that responded to combination therapy. Pharmacological/genomic inhibition of iNOS reversed EMT in MpBC cells, by decreased expression of Zeb1, TGFβ, Snail, Vimentin, and increased expression of E-cadherin and ZO-1 in immunoblotting analysis. MpBC cells with NOS2 knockout acquired an epithelial-like cellular morphology, and this reversal of EMT rendered MpBC cells more sensitive to alpelisib and taxane-chemotherapy. MpBC PDX tumors that responded to combination therapy also exhibited a reversal in EMT, with a decrease in aldehyde dehydrogenase (ALDH1), a CSC marker. Combination therapy also reduced tumor-initiating ability, enhanced chemosensitivity, and improved overall survival in MpBC PDX models. These studies paralleled a phase 1b/2 clinical trial with L-NMMA+taxane chemotherapy in a cohort of anthracycline-refractory MpBC patients (NCT02834403). The clinical benefit rate was 40% (6/15), overall response rate was 20% (3/15), and one patient achieved a pathologic complete response. Relative to baseline tumors, the responder end-of-treatment tumors had undergone reversal of EMT, with decreased expression of iNOS and ALDH1. We find that combining L-NMMA and alpelisib is a novel therapeutic strategy to treat MpBC, and combination therapy is being tested in a first multicenter phase 2 study for patients with MpBC. Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Christoforos Thomas, Wei Qian, Jianying Zhou, Hong Zhao, Xiaoxian Li, Bijan Mahboubi, Adrian Oo, Cho Young-Jae, Baek Kim, Jose Thaiparambil, Maria Florencia Chervo, Roberto Rosato, Camila Ayerbe, Noah Giese, Stacy Moulder, Helen Piwnica-Worms, Funda Meric-Bernstam, Jenny C. Chang. NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3447.