A novel HSP90 inhibitor SL-145 suppresses metastatic triple-negative breast cancer without triggering the heat shock response.

Despite recent advances, there remains a significant unmet need for the development of new targeted therapies for triple-negative breast cancer (TNBC). Although the heat shock protein HSP90 is a promising target, previous inhibitors have had issues during development including undesirable induction of the heat shock response (HSR) and off-target effects leading to toxicity. SL-145 is a novel, rationally-designed C-terminal HSP90 inhibitor that induces apoptosis in TNBC cells via the suppression of oncogenic AKT, MEK/ERK, and JAK2/STAT3 signaling and does not trigger the HSR, in contrast to other inhibitors. In an orthotopic allograft model incorporating breast cancer stem cell-enriched TNBC tumors, SL-145 potently suppressed tumor growth, angiogenesis, and metastases concomitant with dysregulation of the JAK2/STAT3 signaling pathway. Our findings highlight the potential of SL-145 in suppressing metastatic TNBC independent of the HSR.

Investigation of B,C-ring truncated deguelin derivatives as heat shock protein 90 (HSP90) inhibitors for use as anti-breast cancer agents.

On the basis of deguelin, a series of the B,C-ring truncated surrogates with N-substituted amide linkers were investigated as HSP90 inhibitors. The structure activity relationship of the template was studied by incorporating various substitutions on the nitrogen of the amide linker and examining their HIF-1α inhibition. Among them, compound 57 showed potent HIF-1α inhibition and cytotoxicity in triple-negative breast cancer lines in a dose-dependent manner. Compound 57 downregulated expression and phosphorylation of major client proteins of HSP90 including AKT, ERK and STAT3, indicating that its antitumor activity was derived from the inhibition of HSP90 function. The molecular modeling of 57 demonstrated that 57 bound well to the C-terminal ATP-binding pocket in the open conformation of the hHSP90 homodimer with hydrogen bonding and pi-cation interactions. Overall, compound 57 is a potential antitumor agent for triple-negative breast cancer as a HSP90 C-terminal inhibitor.

C-terminal HSP90 inhibitor L80 elicits anti-metastatic effects in triple-negative breast cancer via STAT3 inhibition.

Triple-negative breast cancer (TNBC) is an aggressive heterogeneous disease with a divergent profile. It has an earlier tendency to form metastases and is associated with poor clinical outcomes due to the limited treatment options available. Heat-shock protein (HSP90) represents a potential treatment target as it promotes tumor progression and metastasis by modulating the maturation and stabilization of signal transduction proteins. We sought to investigate the efficacy of the C-terminal HSP90 inhibitor L80 on cell proliferation, breast cancer stem cell (BCSC)-like properties, tumor growth and metastasis. L80 suppressed cell viability and concomitantly inhibited AKT/MEK/ERK/JAK2/STAT3 signaling in TNBC cells but did not induce cytotoxicity in normal cells. L80 effectively targeted BCSC-like traits, together with significant reductions in the CD44high/CD24low-population, ALDH1 activity and mammosphere forming-ability. In support of the in vitro observations, L80 administration caused significant impairment in tumor growth, angiogenesis and distant metastases in an orthotopic allograft model with BCSC-enriched cells in vivo. These phenomena were associated with the suppression of BCSC-like characteristics and STAT3 dysfunction. Our findings highlight properties of the L80 compound that may be useful in suppressing metastatic TNBC.

Flubendazole elicits anti-metastatic effects in triple-negative breast cancer via STAT3 inhibition.

Tumor metastasis remains the cause of 90% of cancer-related deaths. Cancer stem cells (CSC) are thought to be responsible for the aggressive and metastatic nature of triple-negative breast cancers (TNBC), and new therapeutic strategies are being devised to target them. Flubendazole (FLU) is a widely used anthelmintic agent that also exhibits anticancer activity in several cancer types. The aim of this study was to characterize the mechanism of action of FLU on breast cancer stem cell (BCSC)-like properties and metastasis in TNBC. FLU treatment caused a significant induction of apoptosis, accompanied by G2/M phase accumulation, caspase-3/-7 activation and the dysregulation of STAT3 activation in TNBC cells. The latter phenomenon was associated with impairment of cancer stem-like traits, concomitant with a reduction in the CD24low /CD44high , CD24high /CD49fhigh subpopulation, ALDH1 activity and mammosphere formation. The BCSC-enriched populations exhibited enhanced metastasis with higher STAT3 activation, while FLU administration inhibited tumor growth, angiogenesis and lung and liver metastasis, coinciding with decreased MMP-2 and MMP-9 levels in circulating blood. FLU kills not only rapid proliferating tumor cells but also effectively eradicates BCSC-like cells in vitro and in vivo. Our findings warrant further investigation of FLU as a treatment for metastatic TNBC.

Flubendazole overcomes trastuzumab resistance by targeting cancer stem-like properties and HER2 signaling in HER2-positive breast cancer.

Although trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Trastuzumab resistance is a multi-factorial phenomenon thought to arise from the presence of cancer stem cells and interactions between truncated p95HER2 and HER family members. Flubendazole (FLU) is a potent anthelmintic agent with an exceptional safety profile. Evidence also suggests that it can act as an anticancer agent in several cancer cell types. We sought to investigate the effect of FLU on apoptosis, HER2/Akt signaling, breast cancer stem cell (BCSC)-like properties and trastuzumab resistance in HER2-positive breast cancer cells. FLU treatment induced apoptosis, associated with a significant downregulation of truncated p95HER2, phospho-HER2, phospho-HER3 and phospho-Akt levels, as well as suppression of HER2/HER3 hetero-dimerization in both trastuzumab-sensitive and -resistant lines. FLU effectively targeted BCSC-like properties including aldehyde dehydrogenase 1 (ALDH1) expression and the CD44high/CD24low phenotype, concomitant with a suppression of mammosphere-forming ability. FLU administration also caused significant tumor suppression in trastuzumab-resistant xenografts, coinciding with the downregulation of BCSC-related markers and intracellular HER2. These findings highlight the mechanisms of action of FLU in overcoming trastuzumab resistance in breast cancer.

Inhibition of ubiquitin-specific protease 34 (USP34) induces epithelial-mesenchymal transition and promotes stemness in mammary epithelial cells.

Ubiquitin-specific protease 34 (USP34) is a deubiquitinating enzyme that regulates Axin stability and plays a critical role in Wnt/ß-catenin signaling. We sought to investigate the role of USP34 on epithelial-mesenchymal (EMT) induction and its effects on mammary epithelial stem cells. USP34 expression levels were relatively lower in MDA-MB-231 and 4T1 mesenchymal-like cells when compared to epithelial-like cells. Inhibition of USP34 in NMuMG cells induced EMT, as evidenced by the upregulation of EMT markers including N-cadherin, phospho-Smad3, Snail and active-ß-catenin, as well as the downregulation of Axin 1 and E-cadherin. USP34 knockdown (KD) in these cells also resulted in the acquisition of invasive behavior, and promoted stemness as indicated by enhanced mammosphere-forming ability, concomitant with the upregulation of Nanog, Oct4 and Sox2 mRNA expression. Endogenous USP34 expression was observed to be at low levels in virgin mouse mammary glands in vivo. When USP34-KD cells were transplanted into the cleared mammary fat pads (CFP) of mice, these cells reconstituted the mammary gland with ductal tree development within 3months. Our findings suggest a previously unknown role for USP34 in mammary gland development.

Disulfiram suppresses cancer stem-like properties and STAT3 signaling in triple-negative breast cancer cells.

In the presence of copper (Cu), disulfiram (DSF) suppresses properties associated with cancer stem cells (CSCs) in breast cancer, but the mechanism of action is poorly understood. In the present study, we observed that DSF/Cu treatment induced apoptosis, mediated by caspase-3 activation in triple-negative breast cancer (TNBC) cells. DSF/Cu treatment also specifically targeted CSC-like cell populations, marked by the inhibition of ALDH1 activity, the suppression of CD44+/CD24-and CD49f+/CD24 + subpopulations, and the subsequent impairment of mammosphere formation. These effects were functionally associated with a significant impact on the STAT3 signaling pathway, characterized by the downregulation of phospho-STAT3, cyclin D1 and survivin. In an MDA-MB-231-derived xenograft model, DSF administration significantly downregulated ALDH1A1, CD44 and phospho-STAT3 levels. These findings show for the first time that DSF suppresses stem-like properties in TNBC by targeting the STAT3 signaling pathway.

Disulfiram induces anoikis and suppresses lung colonization in triple-negative breast cancer via calpain activation.

Triple-negative breast cancers (TNBC) often exhibit an aggressive phenotype. Disulfiram (DSF) is an approved drug for the treatment of alcohol dependence, but has also been shown to kill TNBC cells in a copper (Cu)-dependent manner. Exactly how this occurs has not been clearly elucidated. We sought to investigate the mechanisms responsible for DSF/Cu-dependent induction of apoptosis and suppression of lung colonization by TNBC cells. DSF/Cu induced anoikis and significantly suppressed cell migration and invasion with negative effects on focal adhesions, coinciding with vimentin breakdown and calpain activation in TNBC cells. In a xenograft tumor model, DSF suppressed tumor growth and lung nodule growth, which was also associated with calpain activation. These findings warrant further investigation of disulfiram as a potential treatment for metastatic TNBC.

Disulfiram targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer.

HER2-positive breast tumors are known to harbor cancer stem-like cell populations and are associated with an aggressive tumor phenotype and poor clinical outcomes. Disulfiram (DSF), an anti-alcoholism drug, is known to elicit cytotoxicity in many cancer cell types in the presence of copper (Cu). The objective of the present study was to investigate the mechanism of action responsible for the induction of apoptosis by DSF/Cu and its effect on cancer stem cell properties in HER2-positive breast cancers in vitro and in vivo. DSF/Cu treatment induced apoptosis, associated with a marked decrease in HER2, truncated p95HER2, phospho-HER2, HER3, phospho-HER3 and phospho-Akt levels, and p27 nuclear accumulation. This was accompanied by the eradication of cancer stem-like populations, concomitant with the suppression of aldehyde dehydrogenase 1 (ALDH1) activity and mammosphere formation. DSF administration resulted in a significant reduction in tumor growth and an enhancement of apoptosis, as well as HER2 intracellular domain (ICD) and ALDH1A1 downregulation. Our results demonstrate that DSF/Cu induces apoptosis and eliminates cancer stem-like cells via the suppression of HER2/Akt signaling, suggesting that DSF may be potentially effective for the treatment of HER2-positive cancers.

MicroRNA-20b inhibits the proliferation, migration and invasion of bladder cancer EJ cells via the targeting of cell cycle regulation and Sp-1-mediated MMP-2 expression.

MicroRNAs (miRs) serve either as oncogenes or tumor-suppressor genes in tumor progression. MicroRNA-20b (miR­20b) is known to be involved with the oncomirs of several types of cancers. However, in the present study we describe how miR-20b inhibits the proliferation, migration and invasion of bladder cancer EJ cells. In the present study, miR-20b was downregulated in bladder cancer cell lines, and its overexpression resulted in a significant reduction in the proliferation of EJ cells. In addition, via a bioinformatics approach, we identified cell cycle-regulated genes that are the putative targets of miR-20b. The transfection of miR-20b into EJ cells induced G1 phase cell cycle arrest via the decreased expression of cyclin D1, CDK2 and CDK6 without affecting another G1 phase cell cycle regulator, cyclin E. The cell cycle inhibitor p21WAF1 was upregulated in the miR-20b transfected cells. Moreover, the enforced expression of miR-20b resulted in impaired wound-healing migration and invasion in the EJ cells. Based on our target prediction analysis of miRs, we confirmed that miR-20b overexpression strongly impedes MMP-2 expression via suppressive activation of the Sp-1 binding motif, an important transcription factor present in the MMP-2 promoter. Herein, we report the novel concept that miR-20b exerts a suppressive effect on both cell cycle-modulated proliferation and MMP-2-mediated migration and invasion in bladder cancer EJ cells.

AKT signaling is involved in fucoidan-induced inhibition of growth and migration of human bladder cancer cells.

We identified a novel mechanism of AKT signaling in the fucoidan-induced proliferation and migration of human urinary 5637 cancer cells. Fucoidan treatment showed a significant growth inhibition followed by G1-phase-associated up-regulation of p21WAF1 expression and suppression of cyclins and CDK expression in 5637 cells. Also, fucoidan treatment induced the activation of AKT signaling, which was inhibited by treatment with wortmannin, a PI3K-specific inhibitor. Blockade of the AKT function reversed the fucoidan-mediated inhibition of cell proliferation, the increased G1-phase-associated p21WAF1 expression, and the reduction of cell-cycle proteins. Moreover, treatment with fucoidan blocked migration and invasion of 5637 cells. This inhibition was attributed to decreased expression of MMP-9, which was mediated by down-regulation of AP-1 and NF-κB binding activity. Furthermore, wortmannin treatment abolished the decreased cell migration and invasion and the inhibition of MMP-9 expression via the suppression of NF-κB and AP-1 in fucoidan-treated cells. Similar results were observed in another bladder cancer T-24 cells treated with fucoidan. Finally, overexpression of the AKT gene inhibited the proliferation, migration and invasion of bladder cancer cells. These data suggest that the activation of AKT signaling is involved in growth inhibition and suppression of the migration and invasion of bladder cancer cells treated with fucoidan.