Metastatic growth in breast cancer (BC) has been proposed as an exclusive property of cancer stem cells (CSCs). However, formal proof of their identity as cells of origin of recurrences at distant sites and the molecular events that may contribute to tumor cell dissemination and metastasis development are yet to be elucidated. In this study, we analyzed a set of patient-derived breast cancer stem cell (BCSC) lines. We found that in vitro BCSCs exhibit a higher chemoresistance and migratory potential when compared with differentiated, nontumorigenic, breast cancer cells (dBCCs). By developing an in vivo metastatic model simulating the disease of patients with early BC, we observed that BCSCs is the only cell population endowed with metastatic potential. Gene-expression profile studies comparing metastagenic and non-metastagenic cells identified TAZ, a transducer of the Hippo pathway and biomechanical cues, as a central mediator of BCSCs metastatic ability involved in their chemoresistance and tumorigenic potential. Overexpression of TAZ in low-expressing dBCCs induced cell transformation and conferred tumorigenicity and migratory activity. Conversely, loss of TAZ in BCSCs severely impaired metastatic colonization and chemoresistance. In clinical data from 99 BC patients, high expression levels of TAZ were associated with shorter disease-free survival in multivariate analysis, thus indicating that TAZ may represent a novel independent negative prognostic factor. Overall, this study designates TAZ as a novel biomarker and a possible therapeutic target for BC.

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

The Hippo signalling is emerging as a tumour suppressor pathway whose function is regulated by an intricate network of intracellular and extracellular cues. Defects in the signal cascade lead to the activation of the Hippo transducers TAZ and YAP. Compelling preclinical evidence showed that TAZ/YAP are often aberrantly engaged in breast cancer (BC), where their hyperactivation culminates into a variety of tumour-promoting functions such as epithelial-to-mesenchymal transition, cancer stem cell generation and therapeutic resistance. Having acquired a more thorough understanding in the biology of TAZ/YAP, and the molecular outputs they elicit, has prompted a first wave of exploratory, clinically-focused analyses aimed at providing initial hints on the prognostic/predictive significance of their expression. In this review, we discuss oncogenic activities linked with TAZ/YAP in BC, and we propose clinical strategies for investigating their role as biomarkers in the clinical setting. Finally, we address the therapeutic potential of TAZ/YAP targeting and the modalities that, in our opinion, should be pursued in order to further study the biological and clinical consequences of their inhibition.

The incidence of colon cancer has increased in developed countries, possibly due to sedentary lifestyle and high caloric diet. Experimental and epidemiological evidence suggests a link between colon cancer development and adipose tissue-derived circulating hormones. Leptin, a pluripotent cytokine secreted by adipocytes, is a key regulator of appetite and energy balance acting in the brain. On the other hand, leptin also controls many physiological and pathological processes in peripheral organs. Recent studies in colon cancer cell lines and human tumors suggested that leptin and its receptor (ObR) are implicated in colon carcinogenesis, and may serve as new biomarkers and pharmacological targets. Here, we explored, for the first time, whether leptin can affect the biology of colorectal tumor stem cells (CTSCs). We found that our previously established and characterized CTSC clones express ObR and respond to leptin with cell proliferation, activation of the extracellular signal-related kinase (ERK)1/2 and AKT signaling pathways, enhanced growth in soft agar, and improved sphere formation associated with E-cadherin overexpression. Moreover, leptin counteracted cytotoxic effects of 5-fluorouracil, a common colon cancer therapeutic agent. These results suggest that obesity and increased leptin levels might promote colorectal cancer by increasing growth and survival of CTSCs.

Recombinant erythropoietin (EPO) analogs [erythropoiesis-stimulating agents (ESA)] are clinically used to treat anemia in patients with cancer receiving chemotherapy. After clinical trials reporting increased adverse events and/or reduced survival in ESA-treated patients, concerns have been raised about the potential role of ESAs in promoting tumor progression, possibly through tumor cell stimulation. However, evidence is lacking on the ability of EPO to directly affect cancer stem-like cells, which are thought to be responsible for tumor progression and relapse. We found that breast cancer stem-like cells (BCSC) isolated from patient tumors express the EPO receptor and respond to EPO treatment with increased proliferation and self-renewal. Importantly, EPO stimulation increased BCSC resistance to chemotherapeutic agents and activated cellular pathways responsible for survival and drug resistance. Specifically, the Akt and ERK pathways were activated in BCSC at early time points following EPO treatment, whereas Bcl-xL levels increased at later times. In vivo, EPO administration counteracted the effects of chemotherapeutic agents on BCSC-derived orthotopic tumor xenografts and promoted metastatic progression both in the presence and in the absence of chemotherapy treatment. Altogether, these results indicate that EPO acts directly on BCSC by activating specific survival pathways, resulting in BCSC protection from chemotherapy and enhanced tumor progression. Cancer Res; 73(21); 6393-400. Ó2013 AACR.

The discovery of inhibitors for oncogenic signalling pathways remains a key focus in modern oncology, based on personalized and targeted therapeutics. Computational drug repurposing via the analysis of FDA-approved drug network is becoming a very effective approach to identify therapeutic opportunities in cancer and other human diseases. Given that gene expression signatures can be associated with specific oncogenic mutations, we tested whether a “reverse” oncogene-specific signature might assist in the computational repositioning of inhibitors of oncogenic pathways. As a proof of principle, we focused on oncogenic PI3K-dependent signalling, a molecular pathway frequently driving cancer progression as well as raising resistance to anticancer-targeted therapies. We show that implementation of “reverse” oncogenic PI3K-dependent transcriptional signatures combined with interrogation of drug networks identified inhibitors of PI3K-dependent signalling among FDA-approved compounds. This led to repositioning of Niclosamide (Niclo) and Pyrvinium Pamoate (PP), two anthelmintic drugs, as inhibitors of oncogenic PI3K-dependent signalling. Niclo inhibited phosphorylation of P70S6K, while PP inhibited phosphorylation of AKT and P70S6K, which are downstream targets of PI3K. Anthelmintics inhibited oncogenic PI3K-dependent gene expression and showed a cytostatic effect in vitro and in mouse mammary gland. Lastly, PP inhibited the growth of breast cancer cells harbouring PI3K mutations. Our data indicate that drug repositioning by network analysis of oncogene-specific transcriptional signatures is an efficient strategy for identifying oncogenic pathway inhibitors among FDA-approved compounds. We propose that PP and Niclo should be further investigated as potential therapeutics for the treatment of tumors or diseases carrying the constitutive activation of the PI3K/P70S6K signalling axis.

We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients.

◥Cancer stem-like cells (CSC) induce aggressive tumor phenotypes such as metastasis formation, which is associated with poor prognosis in triple-negative breast cancer (TNBC). Repurposing of FDA-approved drugs that can eradicate the CSC subcompartment in primary tumors may prevent metastatic disease, thus representing an effective strategy to improve the prognosis of TNBC. Here, we investigated spheroid-forming cells in a metastatic TNBC model. This strategy enabled us to specifically study a population of longlived tumor cells enriched in CSCs, which show stem-like characteristics and induce metastases. To repurpose FDA-approved drugs potentially toxic for CSCs, we focused on pyrvinium pamoate (PP), an anthelmintic drug with documented anticancer activity in preclinical models. PP induced cytotoxic effects in CSCs and prevented metastasis formation. Mechanistically, the cell killing effects of PP were a result of inhibition of lipid anabolism and, more specifically, the impairment of anabolic flux from glucose to cholesterol and fatty acids. CSCs were strongly dependent upon activation of lipid biosynthetic pathways; activation of these pathways exhibited an unfavorable prognostic value in a cohort of breast cancer patients, where it predicted high probability of metastatic dissemination and tumor relapse. Overall, this work describes a new approach to target aggressive CSCs that may substantially improve clinical outcomes for patients with TNBC, who currently lack effective targeted therapeutic options.Significance: These findings provide preclinical evidence that a drug repurposing approach to prevent metastatic disease in TNBC exploits lipid anabolism as a metabolic vulnerability against CSCs in primary tumors.