Tejaswini P. Reddy

Treatment modalities for pulmonary arterial hypertension (PAH) improve quality of life and walk distance. However, none of these therapies alter the structural/functional pulmonary vascular integrity that results in vascular remodeling. PAH smooth muscle cells share biological characteristics with cancer cells, which may be potential therapeutic targets for PAH. We present a case of a patient with connective tissue disease (CTD)‐associated PAH treated on triple therapy who developed metastatic lung adenocarcinoma. While on PAH triple‐therapy, she received a combination of carboplatin, pemetrexed, and pembrolizumab. She eventually had a complete pathologic response, no evidence of cancer recurrence, and significant improvement of PAH/overall clinical status. After discontinuation of neoplastic therapy, her clinical status worsened, she eventually passed away, and lung biopsy findings revealed evidence of severe pulmonary smooth muscle cell hypertrophy and pulmonary veno‐occlusive disease. This report suggests that combined chemotherapy and immunotherapy may influence the efficacy of PAH therapies and improve clinical status.

Utilizing targeted therapies capable of reducing cancer metastasis, targeting chemoresistant and self-renewing cancer stem cells, and augmenting the efficacy of systemic chemo/radiotherapies is vital to minimize cancer-associated mortality. Targeting nitric oxide synthase (NOS), a protein within the tumor microenvironment, has gained interest as a promising therapeutic strategy to reduce metastatic capacity and augment the efficacy of chemo/radiotherapies in various solid malignancies. Our review highlights the influence of nitric oxide (NO) in tumor progression and cancer metastasis, as well as promising preclinical studies that evaluated NOS inhibitors as anticancer therapies. Lastly, we highlight the prospects and outstanding challenges of using NOS inhibitors in the clinical setting.

Purpose: Metaplastic breast cancer (MpBC) is a highly chemoresistant and aggressive breast cancer variant with limited therapeutic options. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis compared to TNBC. We and others discovered two key molecular alterations in MpBC associated with poor overall survival: 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced inducible nitric oxide synthase (iNOS) signaling. Nitric oxide produced from iNOS is able to activate various oncogenic pathways, such as PI3K signaling. In MpBC, both PI3K and NOS signaling pathways may synergistically work to enhance chemoresistance. We propose to evaluate 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 (Hs578T, BT549, SUM159) and MpBC Patient-Derived Xenograft (PDX) models were used in this study. Cell viability was determined with SRB Assay and the combination index was evaluated using Chou-Talalay Method. Flow cytometry was conducted to evaluate cellular apoptosis and cell cycle distribution analysis. In vivo efficacy of NOS and PI3K inhibition was assessed using MpBC PDX models. Transcriptional analysis of PDX tumors treated with vehicle control, single-agent (L-NMMA or alpelisib), or combination therapy (L-NMMA+alpelisib) was conducted to reveal top enriched pathways. Immunoblotting and immunofluorescence analyses of treated MpBC cell lines and PDX tissues were performed. Results: Combination of L-NMMA and alpelisib synergistically reduced cellular proliferation (CICitation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Bijan Mahboubi, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Roberto Rosato, Jenny C. Chang. NOS inhibition augments PI3K inhibitor-induced DNA damage and mesenchymal-to-epithelial transition in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2658.

Background: Metaplastic breast cancer (MpBC) is a therapeutically chemoresistant, aggressive, and heterogeneous breast cancer variant accounting for <5% of all breast cancers. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis and decreased survival compared to TNBC. Despite its chemorefractory nature, the current mainstay of treatment for MpBC is surgery and systemic chemotherapy. Common molecular alterations found in MpBC associated with poor prognosis and worse overall survival include 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced production of nitric oxide via inducible nitric oxide synthase (iNOS). In MpBC, both the PI3K and NOS signaling pathways may synergistically work together to enhance chemoresistance. We propose that combined inhibition of PI3K and iNOS will enhance the efficacy of taxane-based chemotherapy in MpBC. Methods: For in vitro and in vivo studies, we used MpBC cell lines (Hs578T and BT549) and TNBC/MpBC Patient-Derived Xenograft (PDX) models, respectively. For all studies, we used pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA, L), PI3K inhibitor alpelisib (A), and docetaxel (D). Immunohistochemistry (IHC), Western Blotting (WB), Cell Proliferation Assays, Flow Cytometry (to evaluate cell death and cell cycle distribution analysis), and HIV reverse transcriptase-based dNTP assay to quantify dNTPs were performed. For in vivo studies, five MpBC PDX models were implanted into the mammary fat pad of NSG mice and they received single therapy (vehicle control, L, A, D), dual therapy (D+A, D+L), or triple combination therapy (D+A+L). Tumor volumes were recorded twice weekly. Results: 66% (4/6) MpBC and 33% (7/21) TNBC PDX models had double-positive IHC staining of both iNOS and p-Akt (Ser473), supporting the concept that both signaling pathways are typically activated in MpBC tumors, relative to non-metaplastic TNBC tumors. Apoptosis and cell proliferation analysis found that MpBC cell lines treated with triple-combination (D+A+L) had an increased number of apoptotic cells and decreased cell proliferation relative to MpBC cells treated with dual combination (L+A), or single treatment (vehicle, D, A, or L). Cell cycle distribution analysis of treated MpBC cells found that in a time-dependent manner, there was a substantial decrease in the % of MpBC cells in S-phase and an increase in the % cells in G2/M cell cycle arrest due to dual and triple combination. This result was supported by dNTP quantification analysis revealing that combined PI3K and NOS inhibition induced greater nucleotide depletion within 8 hours of treatment relative to single treatment in MpBC cells. WB analysis revealed that dual/triple combination therapy in MpBC cells resulted in an enhanced DNA damage response signaling relative to single treatment, as indicated with increased expression of γ-H2AX, p-Chk1, p-Chk2, p-P53 (Ser15 and 20), and p21. Pro-survival PI3K signaling pathway was activated in response to docetaxel treatment alone in MpBC cells, but its activation was significantly reduced when docetaxel was coupled with PI3K and NOS inhibition. In vivo studies revealed that triple combination therapy significantly reduced tumor volume and improved survival proportions compared to dual/single therapy and vehicle control. Conclusions: The present data suggest that combined PI3K and NOS inhibition enhances docetaxel-mediated DNA damage by depleting nucleotide pools, leading to enhanced DNA damage response, growth arrest, and apoptosis. Ongoing studies are investigating how docetaxel coupled with PI3K and NOS inhibition influences DNA repair signaling and MpBC metastatic capacity. The addition of PI3K and NOS inhibitors to taxane-based chemotherapy may be a novel therapeutic strategy for aggressive MpBCs. Citation Format: Tejaswini P Reddy, Bijan Mahboubi, Roberto R. Rosato, Liliana Guzman-Rojas, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Jenny C. Chang. Combined PI3K and NOS inhibition enhances efficacy of taxane-based chemotherapy in metaplastic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-04.

Metastatic triple‐negative breast cancer (mTNBC) patients tend to have a poor overall survival. The primary goals of treatment focus on palliation of symptoms and improvement in overall survival (OS). Single‐agent sequential chemotherapy with anthracycline or taxane has remained the cornerstone of treatment for many years. The FDA has approved newer agents such as poly‐adenosine diphosphate‐ribose polymerase (PARP) inhibitors upfront in germline BRCA (gBRCA) 1/2 mutation carriers; atezolizumab and nab‐paclitaxel combination frontline in patients with PD‐L1 expression > 1%; and sacituzumab govitecan (IMMU‐132), an antibody‐drug conjugate in heavily pretreated mTNBC patients.

Background: The human epidermal growth factor receptor (HER) family, notably EGFR, is overexpressed in most triple-negative breast cancer (TNBC) cases and provides cancer cells with compensatory signals that greatly contribute to the survival and development of resistance in response to therapy. This study investigated the effects of Pan-HER (Symphogen, Ballerup, Denmark), a novel mixture of six monoclonal antibodies directed against members of the HER family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs). Methods: Fifteen low passage TNBC PDX tumor samples were transferred into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 100–200 mm³, mice were randomized (n ≥ 6 per group) and treated following three 1-week cycles consisting of three times/week intraperitoneal (IP) injection of either formulation buffer (vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for Western blot, RNA, and immunohistochemistry analyses. Results: All 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor downmodulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways AKT and ERK. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation. Conclusions: The marked effect of Pan-HER in numerous HER family-dependent TNBC PDX models justifies further studies of Pan-HER in TNBC clinical trials as a potential therapeutic option.

Sorafenib, a broad tyrosine kinase inhibitor, is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC) but provides limited survival benefits. Recently, immunotherapy has emerged as a promising treatment strategy, but its role remains unclear in HCCs, which are associated with decreased cytotoxic CD8⁺ T‐lymphocyte infiltration in both murine and human tumors. Moreover, in mouse models after sorafenib treatment intratumoral hypoxia is increased and may fuel evasive resistance. Using orthotopic HCC models, we now show that increased hypoxia after sorafenib treatment promotes immunosuppression, characterized by increased intratumoral expression of the immune checkpoint inhibitor programmed death ligand‐1 and accumulation of T‐regulatory cells and M2‐type macrophages. We also show that the recruitment of immunosuppressive cells is mediated in part by hypoxia‐induced up‐regulation of stromal cell–derived 1 alpha. Inhibition of the stromal cell–derived 1 alpha receptor (C‐X‐C receptor type 4 or CXCR4) using AMD3100 prevented the polarization toward an immunosuppressive microenvironment after sorafenib treatment, inhibited tumor growth, reduced lung metastasis, and improved survival. However, the combination of AMD3100 and sorafenib did not significantly change cytotoxic CD8⁺ T‐lymphocyte infiltration into HCC tumors and did not modify their activation status. In separate experiments, antibody blockade of the programmed death ligand‐1 receptor programmed death receptor‐1 (PD‐1) showed antitumor effects in treatment‐naive tumors in orthotopic (grafted and genetically engineered) models of HCC. However, anti‐PD‐1 antibody treatment had additional antitumor activity only when combined with sorafenib and AMD3100 and not when combined with sorafenib alone. Conclusion: Anti‐PD‐1 treatment can boost antitumor immune responses in HCC models; when used in combination with sorafenib, anti‐PD‐1 immunotherapy shows efficacy only with concomitant targeting of the hypoxic and immunosuppressive microenvironment with agents such as CXCR4 inhibitors. (Hepatology 2015;61:1591–1602)

Leptin and oestradiol have overlapping functions in energy homeostasis and fertility, and receptors for these hormones are localised in the same hypothalamic regions. Although, historically, it was assumed that mammalian adult neurogenesis was confined to the olfactory bulbs and the hippocampus, recent research has found new neurones in the male rodent hypothalamus. Furthermore, some of these new neurones are leptin‐sensitive and affected by diet. In the present study, we tested the hypothesis that diet and hormonal status modulate hypothalamic neurogenesis in the adult female mouse. Adult mice were ovariectomised and implanted with capsules containing oestradiol (E₂) or oil. Within each group, mice were fed a high‐fat diet (HFD) or maintained on standard chow (STND). All animals were administered i.c.v. 5‐bromo‐2′‐deoxyuridine (BrdU) for 9 days and sacrificed 34 days later after an injection of leptin to induce phosphorylation of signal transducer of activation and transcription 3 (pSTAT3). Brain tissue was immunohistochemically labelled for BrdU (newly born cells), Hu (neuronal marker) and pSTAT3 (leptin sensitive). Although mice on a HFD became obese, oestradiol protected against obesity. There was a strong interaction between diet and hormone on new cells (BrdU+) in the arcuate, ventromedial hypothalamus and dorsomedial hypothalamus. HFD increased the number of new cells, whereas E₂ inhibited this effect. Conversely, E₂ increased the number of new cells in mice on a STND diet in all hypothalamic regions studied. Although the total number of new leptin‐sensitive neurones (BrdU‐Hu‐pSTAT3) found in the hypothalamus was low, HFD increased these new cells in the arcuate, whereas E₂ attenuated this induction. These results suggest that adult neurogenesis in the hypothalamic neurogenic niche is modulated by diet and hormonal status and is related to energy homeostasis in female mice.