One undisputed milestone of traditional oncology is neoplastic progression, which consists of a progressive selection of dedifferentiated cells driven by a chance sequence of genetic mutations. Recently it has been demonstrated that the overexpression of well-defined transcription factors reprograms somatic cells to the pluripotent stem status. The demonstration raises crucial questions as to whether and to what extent this reprogramming contributes to tumorigenesis, and whether the epigenetic changes involved in it are reversible. Here, we show for the first time that a tumor produced in vivo by a chemical carcinogen is the product of the interaction between neoplastic progression and reprogramming. The experimental model employed the prototype of ascites tumors, the Yoshida AH130 hepatoma and other neoplasias, including human melanoma. AH130 hepatoma was started in the liver by the carcinogen o-aminoazotoluene. This compound binds to and abolishes the p53 protein, producing a genomic instability that promotes both the neoplastic progression and the hepatoma reprogramming. Eventually this tumor contained 100% CD133(+) elements and pO(2)-dependent percentages of the three embryonic transcription factors Nanog, Klf4 and c-Myc. Once transferred into aerobic cultures, the minor cellular fraction expressing this triad generates various types of adherent cells, which are progressively substituted by non-tumorigenic elements committed to fibromuscular, neuronal and glial differentiation. This reprogramming appears to be accomplished stepwise, with the assembly of the triad into a sophisticated transcriptional, oxygen-dependent circuitry, in which Nanog and Klf4 antagonistically regulate c-Myc, and hence, cell hypoxia survival and cell cycle activation.

We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state. Experiments were performed in vivo and in vitro using CSCs obtained from various anaplastic tumors: human melanoma, leukemia, and rat hepatoma cells. Pretreatment of melanoma CSCs with pyruvate significantly reduces their self-renewal in vitro and tumorigenicity in vivo. The metabolic network underlying the cytotoxic effect of the physiological factors was thoroughly defined, principally using AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem stage. This network, based on a tight integration of aerobic glycolysis, cellular redox state, and folate metabolism, is centered on the cellular NADP/NADPH ratio that controls the redox pathway of folate utilization in purine synthesis. On the whole, this study indicates that pyruvate, FH4, and glutamine display anticancer activity, because CSCs are committed to survive and maintain their stemness in hypoxia. When CSC need to differentiate and proliferate, they shift from anaerobic to aerobic status, and the few mitochondria available makes them susceptible to the injury of the above physiological factors. This vulnerability might be exploited for novel therapeutic treatments.

Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

Breast cancer stem cells (BCSC) have been identified in breast carcinoma as CD44 C /CD24 ¡/low cells, which display tumorigenic activity and have the ability to self-renew, differentiate and metastasize. Previous studies showed that extracellular HSP90 (eHSP90) participates in the invasion and metastatic processes of various cancers including breast cancer. Here, we show for the first time that eHSP90 is over-expressed in mammosphere cultures that are derived from the MDA-MB-231, MDA-MB-453 and MCF-7 breast cancer cell lines. These mammospheres are highly enriched in cells of the CD44 C /CD24 ¡/low BCSC phenotype and additionally show high expression of the BCSC markers CD49f and Sox2. Thus our results indicate that eHSP90 represents a potential novel BCSC marker. Moreover, we present evidence that eHSP90 is functionally involved in BCSC activity in vitro and in vivo. Selective neutralization of eHSP90, using the monoclonal antibody mAb 4C5, has the capacity to inhibit stem cell activity in vitro because the formation of mammosphere-derived colonies is dramatically reduced in its presence. In vivo, the treatment of mice with mAb4C5 using a prophylactic protocol, significantly inhibited the primary growth of MDA-MB-231 and mammosphere-derived tumors. More importantly, administration of this antibody in a therapeutic protocol caused a statistically significant regression of established tumors derived from MDA-MB-231 originating mammospheres. Tumor regression was even greater when mAb 4C5 was administered in combination with paclitaxel. Overall, our findings implicate eHSP90 as a potential novel BCSC biomarker. Moreover they show that eHSP90 participates in BCSC-derived primary tumor growth. Finally, we provide additional support for the possible therapeutic value of mAb4C5 in the treatment of breast cancer.Abbreviations: ALDH1, aldehyde dehydrogenase 1; BCSC, breast cancer stem cells; eHSP90, extracellular heat shock protein 90; ER, estrogen receptor; EGF, epidermal growth factor; FGF, fibroblast growth factor; MMP-2, matrix metalloproteinase 2