Proguanil Suppresses Breast Tumor Growth In Vitro and In Vivo by Inducing Apoptosis via Mitochondrial Dysfunction.

Breast cancer, ranking as the second leading cause of female cancer-related deaths in the U.S., demands the exploration of innovative treatments. Repurposing FDA-approved drugs emerges as an expedited and cost-effective strategy. Our study centered on proguanil, an antimalarial drug, reveals notable anti-proliferative effects on diverse breast cancer cell lines, including those derived from patients. Proguanil-induced apoptosis was associated with a substantial increase in reactive oxygen species (ROS) production, leading to reduced mitochondrial membrane potential, respiration, and ATP production. Proguanil treatment upregulated apoptotic markers (Bax, p-H2AX, cleaved-caspase 3, 9, cleaved PARP) and downregulated anti-apoptotic proteins (bcl-2, survivin) in breast cancer cell lines. In female Balb/c mice implanted with 4T1 breast tumors, daily oral administration of 20 mg/kg proguanil suppressed tumor enlargement by 55%. Western blot analyses of proguanil-treated tumors supported the in vitro findings, demonstrating increased levels of p-H2AX, Bax, c-PARP, and c-caspase3 as compared to controls. Our results collectively highlight proguanil's anticancer efficacy in vitro and in vivo in breast cancer, prompting further consideration for clinical investigations.

The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression.

Human prostate cancer can result from chromosomal rearrangements that lead to aberrant ETS gene expression. The mechanisms that lead to fusion-independent ETS factor upregulation and prostate oncogenesis remain relatively unknown. Here, we show that two neighboring transcription factors, Capicua (CIC) and ETS2 repressor factor (ERF), which are co-deleted in human prostate tumors can drive prostate oncogenesis. Concurrent CIC and ERF loss commonly occur through focal genomic deletions at chromosome 19q13.2. Mechanistically, CIC and ERF co-bind the proximal regulatory element and mutually repress the ETS transcription factor, ETV1. Targeting ETV1 in CIC and ERF-deficient prostate cancer limits tumor growth. Thus, we have uncovered a fusion-independent mode of ETS transcriptional activation defined by concurrent loss of CIC and ERF.

Capicua suppresses YAP1 to limit tumorigenesis and maintain drug sensitivity in human cancer.

Inactivation of Capicua (CIC) or upregulation of yes-associated protein 1, YAP1, leads to broad RAS-RAF-MEK-ERK inhibitor resistance and tumor progression in multiple human cancers. Despite these shared malignant phenotypes, it remains unclear whether CIC and YAP1 are mechanistically linked. Here, we show that the ERK-regulated transcription factor CIC can directly repress YAP1 expression through non-consensus GGAAGGAA DNA-binding motifs in a proximal YAP1 regulatory element. Through binding at GGAA repeats, CIC regulates YAP1 transcriptional output in both normal and human cancer cells. Silencing YAP1 in CIC-deficient cells restores MAPK inhibitor sensitivity and suppresses tumor growth. Thus, we uncover a molecular link between the MAPK-ERK effector CIC and YAP1 in human cells and established YAP inhibition as a strategy to target CIC-deficient cancers.

Atovaquone Suppresses Triple-Negative Breast Tumor Growth by Reducing Immune-Suppressive Cells.

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-ß and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-ß, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-ß and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.

Atovaquone Suppresses the Growth of Metastatic Triple-Negative Breast Tumors in Lungs and Brain by Inhibiting Integrin/FAK Signaling Axis.

Triple-negative breast cancer (TNBC) is considered to be the most aggressive and malignant neoplasm and is highly metastatic in nature. In the current study, we investigated the anti-metastatic potential of atovaquone, a protozoal drug prescribed for Pneumocystis pneumonia. We showed that atovaquone induced apoptosis and reduced the survival of several aggressive metastatic TNBC cell lines including metastatic patient-derived cells by reducing the expression of integrin α6, integrin ß4, FAK, Src, and Vimentin. In order to study the efficacy of atovaquone in suppressing metastasized breast tumor cells in brain and lungs, we performed three in vivo experiments. We demonstrated that oral administration of 50 mg/kg of atovaquone suppressed MDA-MB-231 breast tumor growth by 90% in lungs in an intravenous metastatic tumor model. Anti-metastatic effect of atovaquone was further determined by intracardiac injection of 4T1-luc breast tumor cells into the left ventricle of mouse heart. Our results showed that atovaquone treatment suppressed the growth of metastatic tumors in lungs, liver and brain by 70%, 50% and 30% respectively. In an intracranial model, the growth of HCC1806-luc brain tumors in atovaquone treated mice was about 55% less than that of control. Taken together, our results indicate the anti-metastatic effects of atovaquone in vitro and in vivo in various breast tumor metastasis models.

Free Radicals as a Double-Edged Sword: The Cancer Preventive and Therapeutic Roles of Curcumin.

Free radicals, generally composed of reactive oxygen species (ROS) and reactive nitrogen species (RNS), are generated in the body by various endogenous and exogenous systems. The overproduction of free radicals is known to cause several chronic diseases including cancer. However, increased production of free radicals by chemotherapeutic drugs is also associated with apoptosis in cancer cells, indicating the dual nature of free radicals. Among various natural compounds, curcumin manifests as an antioxidant in normal cells that helps in the prevention of carcinogenesis. It also acts as a prooxidant in cancer cells and is associated with inducing apoptosis. Curcumin quenches free radicals, induces antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), and upregulates antioxidative protein markers-Nrf2 and HO-1 that lead to the suppression of cellular oxidative stress. In cancer cells, curcumin aggressively increases ROS that results in DNA damage and subsequently cancer cell death. It also sensitizes drug-resistant cancer cells and increases the anticancer effects of chemotherapeutic drugs. Thus, curcumin shows beneficial effects in prevention, treatment and chemosensitization of cancer cells. In this review, we will discuss the dual role of free radicals as well as the chemopreventive and chemotherapeutic effects of curcumin and its analogues against cancer.

Low Dose of Penfluridol Inhibits VEGF-Induced Angiogenesis.

: Metastasis is considered a major burden in cancer, being responsible for more than 90% of cancer-related deaths. Tumor angiogenesis is one of the main processes that lead to tumor metastasis. Penfluridol is a classic and commonly used antipsychotic drug, which has a great ability to cross the blood-brain barrier. Recent studies have revealed that penfluridol has significant anti-cancer activity in diverse tumors, such as metastatic breast cancer and glioblastoma. Here, we aim to identify the effect of low doses of penfluridol on tumor microenvironment and compare it with its effect on tumor cells. Although low concentration of penfluridol was not toxic for endothelial cells, it blocked angiogenesis in vitro and in vivo. In vitro, penfluridol inhibited VEGF-induced primary endothelial cell migration and tube formation, and in vivo, it blocked VEGF- and FGF-induced angiogenesis in the matrigel plug assay. VEGF-induced VEGFR2 phosphorylation and the downstream p38 and ERK signaling pathways were not affected in endothelial cells, although VEGF-induced Src and Akt activation were abrogated by penfluridol treatment. When cancer cells were treated with the same low concentration of penfluridol, basal Src activation levels were mildly impaired, thus impacting their cell migration and wound healing efficiency. The potential of cancer-induced paracrine effect on endothelial cells was explored, although that did not seem to be a player for angiogenesis. Overall, our data demonstrates that low penfluridol levels, similar to the ones clinically used for anti-psychotic conditions, suppress angiogenic efficiency in the tumor microenvironment.

Cancer cells stemness: A doorstep to targeted therapy.

Recent advances in research on cancer have led to understand the pathogenesis of cancer and development of new anticancer drugs. Despite of these advancements, many tumors have been found to recur, undergo metastasis and develop resistance to therapy. Accumulated evidences suggest that small population of cancer cells known as cancer stem cells (CSC) are responsible for reconstitution and propagation of the disease. CSCs possess the ability to self-renew, differentiate and proliferate like normal stem cells. CSCs also appear to have resistance to anti-cancer therapies and subsequent relapse. The underlying stemness properties of the CSCs are reliant on multiple molecular targets such as signaling pathways, cell surface molecules, tumor microenvironment, apoptotic pathways, microRNA, stem cell differentiation, and drug resistance markers. Thus an effective therapeutic strategy relies on targeting CSCs to overcome the possible tumor relapse and chemoresistance. The targeted inhibition of these stem cell biomarkers is one of the promising approaches to eliminate cancer stemness. This review article summarizes possible targets of cancer cell stemness for the complete treatment of cancer.

Role of Phytochemicals in Cancer Prevention.

The use of synthetic, natural, or biological agents to minimize the occurrence of cancer in healthy individuals is defined as cancer chemoprevention. Chemopreventive agents inhibit the development of cancer either by impeding DNA damage, which leads to malignancy or by reversing or blocking the division of premalignant cells with DNA damage. The benefit of this approach has been demonstrated in clinical trials of breast, prostate, and colon cancer. The continuous increase in cancer cases, failure of conventional chemotherapies to control cancer, and excessive toxicity of chemotherapies clearly demand an alternative approach. The first trial to show benefit of chemoprevention was undertaken in breast cancer patients with the use of tamoxifen, which demonstrated a significant decrease in invasive breast cancer. The success of using chemopreventive agents for protecting the high risk populations from cancer indicates that the strategy is rational and promising. Dietary components such as capsaicin, cucurbitacin B, isoflavones, catechins, lycopenes, benzyl isothiocyanate, phenethyl isothiocyanate, and piperlongumine have demonstrated inhibitory effects on cancer cells indicating that they may serve as chemopreventive agents. In this review, we have addressed the mechanism of chemopreventive and anticancer effects of several natural agents.

Atovaquone: An Antiprotozoal Drug Suppresses Primary and Resistant Breast Tumor Growth by Inhibiting HER2/ß-Catenin Signaling.

Breast cancer is the second leading cause of cancer-related mortality in women. In the current study, we evaluated the anticancer effects of an antiprotozoal drug, atovaquone, against several breast cancer cell lines. Our results showed that atovaquone treatment induced apoptosis and inhibited the growth of all the breast cancer cell lines tested, including several patient-derived cells. In addition, atovaquone treatment significantly reduced the expression of HER2, ß-catenin, and its downstream molecules such as pGSK-3ß, TCF-4, cyclin D1, and c-Myc in vitro Efficacy of atovaquone was further evaluated in an in vivo tumor model by orthotropic implantation of two highly aggressive 4T1 and CI66 breast cancer cells in the mammary fat pad of female mice. Our results demonstrated that oral administration of atovaquone suppressed the growth of CI66 and 4T1 tumors by 70% and 60%, respectively. Paclitaxel is the first-line chemotherapeutic agent for metastatic breast cancer. We demonstrate that atovaquone administration suppressed the growth of 4T1 paclitaxel-resistant tumors by 40%. Tumors from atovaquone-treated mice exhibited reduced HER2, ß-catenin, and c-Myc levels alongside an increase in apoptosis in all the three tumor models when analyzed by Western blotting, IHC, and TUNEL assay. Taken together, our results indicate that atovaquone effectively reduces the growth of primary and paclitaxel-resistant breast tumors. Atovaquone is already in the clinics with high safety and tolerability profile. Therefore, the findings from our studies will potentially prompt further clinical investigation into repurposing atovaquone for the treatment of patients with advanced breast cancer.

Penfluridol overcomes paclitaxel resistance in metastatic breast cancer.

Paclitaxel is a first line chemotherapeutic agent for the patients with metastatic breast cancer. But inherited or acquired resistance to paclitaxel leads to poor response rates in a majority of these patients. To identify mechanisms of paclitaxel resistance, we developed paclitaxel resistant breast cancer cell lines, MCF-7 and 4T1 by continuous exposure to paclitaxel for several months. Western blot analysis showed increased expression of HER2 and ß-catenin pathway in resistant cell lines as compared to parent cells. Hence, we hypothesized that HER2/ß-catenin mediates paclitaxel resistance in breast cancer and suppression of HER2/ß-catenin signaling could overcome paclitaxel resistance. Our data showed that penfluridol (PFL) treatment significantly reduced the survival of paclitaxel-resistant cells. Western blot analysis revealed that PFL treatment suppressed HER2, as well as, ß-catenin pathway. In vivo data confirmed that PFL significantly potentiated tumor growth suppressive effects of paclitaxel in an orthotropic breast cancer model. In addition, tumors from paclitaxel and PFL-treated mice showed reduced HER2 and ß-catenin expression, along with increased apoptosis. Taken together our results demonstrate a novel role of HER2/ß-catenin in paclitaxel resistance and open up new avenues for application of PFL as a therapeutic option for overcoming paclitaxel resistance.

HER2-mediated GLI2 stabilization promotes anoikis resistance and metastasis of breast cancer cells.

Breast cancer metastasis is a multi-step process and requires cells to overcome anoikis. Anoikis is defined as cell-death that occurs due to loss of cell adhesion. During the course of cancer progression, tumor cells acquire resistance to anoikis. However, mechanisms of anoikis resistance are not clear. Human epidermal growth receptor 2 (HER2) overexpressing breast tumors are known to be highly aggressive and metastatic. The mechanisms correlating HER2 with metastasis are poorly understood. We observed increased anoikis resistance in HER2 overexpressing breast cancer cells. In addition, we identified that HER2 overexpression was also associated with increased sonic hedgehog (SHH) signaling especially GLI2, and that inhibition of SHH pathway suppressed anoikis resistance. GSK3ß is known to facilitate proteasome-mediated degradation of GLI2. Moreover, we observed that silencing of GLI2 resulted in reduced migration and invasion of HER2 overexpressing cells. Anoikis resistant HER2 overexpressing cells also showed increased rate and extent of metastasis in vivo, as compared to wild type anoikis resistant cells. Taken together, this study indicates a novel role of HER2/GSK3ß/GLI2 axis in anoikis resistance and metastasis, and that GLI2 could be a potential target for anti-cancer therapies.

AKR1C3 Inhibitor KV-37 Exhibits Antineoplastic Effects and Potentiates Enzalutamide in Combination Therapy in Prostate Adenocarcinoma Cells.

Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 17 ß-hydroxysteroid dehydrogenase, is responsible for intratumoral androgen biosynthesis, contributing to the development of castration-resistant prostate cancer (CRPC) and eventual chemotherapeutic failure. Significant upregulation of AKR1C3 is observed in CRPC patient samples and derived CRPC cell lines. As AKR1C3 is a downstream steroidogenic enzyme synthesizing intratumoral testosterone (T) and 5α-dihydrotestosterone (DHT), the enzyme represents a promising therapeutic target to manage CRPC and combat the emergence of resistance to clinically employed androgen deprivation therapy. Herein, we demonstrate the antineoplastic activity of a potent, isoform-selective and hydrolytically stable AKR1C3 inhibitor (E)-3-(4-(3-methylbut-2-en-1-yl)-3-(3-phenylpropanamido)phenyl)acrylic acid (KV-37), which reduces prostate cancer cell growth in vitro and in vivo and sensitizes CRPC cell lines (22Rv1 and LNCaP1C3) toward the antitumor effects of enzalutamide. Crucially, KV-37 does not induce toxicity in nonmalignant WPMY-1 prostate cells nor does it induce weight loss in mouse xenografts. Moreover, KV-37 reduces androgen receptor (AR) transactivation and prostate-specific antigen expression levels in CRPC cell lines indicative of a therapeutic effect in prostate cancer. Combination studies of KV-37 with enzalutamide reveal a very high degree of synergistic drug interaction that induces significant reduction in prostate cancer cell viability via apoptosis, resulting in >200-fold potentiation of enzalutamide action in drug-resistant 22Rv1 cells. These results demonstrate a promising therapeutic strategy for the treatment of drug-resistant CRPC that invariably develops in prostate cancer patients following initial treatment with AR antagonists such as enzalutamide. Mol Cancer Ther; 17(9); 1833-45. ©2018 AACR.

Role of Forkhead Box Class O proteins in cancer progression and metastasis.

It is now widely accepted that several gene alterations including transcription factors are critically involved in cancer progression and metastasis. Forkhead Box Class O proteins (FoxOs) including FoxO1/FKHR, FoxO3/FKHRL1, FoxO4/AFX and FoxO6 transcription factors are known to play key roles in proliferation, apoptosis, metastasis, cell metabolism, aging and cancer biology through their phosphorylation, ubiquitination, acetylation and methylation. Though FoxOs are proved to be mainly regulated by upstream phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt signaling pathway, the role of FoxOs in cancer progression and metastasis still remains unclear so far. Thus, with previous experimental evidences, the present review discussed the role of FoxOs in association with metastasis related molecules including cannabinoid receptor 1 (CNR1), Cdc25A/Cdk2, Src, serum and glucocorticoid inducible kinases (SGKs), CXCR4, E-cadherin, annexin A8 (ANXA8), Zinc finger E-box-binding homeobox 2 (ZEB2), human epidermal growth factor receptor 2 (HER2) and mRNAs such as miR-182, miR-135b, miR-499-5p, miR-1274a, miR-150, miR-34b/c and miR-622, subsequently analyzed the molecular mechanism of some natural compounds targeting FoxOs and finally suggested future research directions in cancer progression and metastasis.

Selective AKR1C3 Inhibitors Potentiate Chemotherapeutic Activity in Multiple Acute Myeloid Leukemia (AML) Cell Lines.

We report the design, synthesis, and evaluation of potent and selective inhibitors of aldo-keto reductase 1C3 (AKR1C3), an important enzyme in the regulatory pathway controlling proliferation, differentiation, and apoptosis in myeloid cells. Combination treatment with the nontoxic AKR1C3 inhibitors and etoposide or daunorubicin in acute myeloid leukemia cell lines, elicits a potent adjuvant effect, potentiating the cytotoxicity of etoposide by up to 6.25-fold and the cytotoxicity of daunorubicin by >10-fold. The results validate AKR1C3 inhibition as a common adjuvant target across multiple AML subtypes. These compounds in coadministration with chemotherapeutics in clinical use enhance therapeutic index and may avail chemotherapy as a treatment option to the pediatric and geriatric population currently unable to tolerate the side effects of cancer drug regimens.