Ovarian cancer stem cells : A target for oncological therapy

According to numerous studies, failures in treatment of ovarian cancer, i.e., a relapse and metastases, result from a small population of cancer stem cells (CSCs). They may also be responsible for tumor initiation. Cancer stem cells are resistant to chemoand radiotherapy. Eradication of CSCs may involve the application of salinomycin, metformin and Clostridium perfringens; the effect of anti-angiogenic factors remains controversial. Salinomycin is an antibiotic isolated from Streptomyces albus bacteria. Its CSC-eradicating effect has been demonstrated both in ovarian cancer cell lines and in women with breast cancer. Clostridium perfringens enterotoxin (CPE) has been demonstrated to destroy CSCs in ovarian cancer both in vivo and in vitro. Metformin, apart from its hypoglycemic effect, reduces the CSC population and inhibits the proliferation of neoplastic cells and angiogenesis. Cancer stem cells with expression of VEGFR1+ have been described as affecting circulating cancer cells and influencing the formation of metastases. Both positive and negative effects of anti-angiogenic therapy on the CSC population have been documented.

Among all malignant gynecological tumors, ovarian cancer represents the leading cause of death.The majority of patients are diagnosed with advanced stages.Standard treatment for ovarian cancer is primary maximum cytoreductive surgery followed by chemotherapy with carboplatin and paclitaxel.After initial debulking and first line chemotherapy, around 50-70% of patients reach full clinical response.However, in 80% of patients this is followed by a relapse of the disease, the cause of therapeutic failure. 1,2ccording to numerous studies, the principal cause of the cancer relapse and metastases stems from a small cell population resistant to chemo-and radiotherapy, comprising less than 2-5% of tumor mass and involving cancer stem cells (CSCs).][5][6] Cancer stem cells manifest the capacity of self-renewal, DNA repair, persisting in the G1 or G0 cell cycle phases as inactive dormant cells and asymmetric cell division.They do not undergo apoptosis and they manifest overexpression of ABC genes, which is linked to their resistance to cytostatic drugs.Control of their self-replacement is associated in principle with numerous signaling pathways, including Notch, Sonic hedgehog (Shh) and Wnt. 2,7,8][11] In 2010, CSCs were isolated and identified in ovarian cancer. 10In 2014, in in vivo and in vitro conditions, cisplatin was demonstrated to inhibit both the formation of metastases and the invasion of CICs manifesting the marker of CD/44 + /CD117 + , but only those with overexpression of CXCR4.Thus, cisplatin inhibited the activity of the SDF-1/CXCR4 axis (the factor originating from stromal cells and its ligand). 12sing an animal model, Abubaker et al. demonstrated that short-term application of chemotherapy (cisplatin and paclitaxel) enriched the cell population with high expression of Oct-4 and CD117. 13Similar experiments were performed by Zhou et al. 14 Following the administration of cisplatin, the dormant cells of human ovarian cancer manifested an augmented expression of cells with Oct-4, nestin, CD-117, and CD44 markers, proving the resistance of this stem cell fraction to cisplatin.
Both the CSC population with its typical markers and the signaling pathways that CSCs take advantage of are thought to provide potential targets for therapy. 7,8,13ccording to Massard et al., there are 2 options for eradicating CSCs: one involves the induction of their differentiation, upon which they lose their capacity of selfreplacement (retinoic acid demonstrated such an ability); the other one involves the elimination of CSCs by targeted inhibitors of signaling pathways. 3elow, we outline the manners of CSC eradication using salinomycin, metformin and enterotoxin of Clostridium perfringens, and we discuss the controversial effects of anti-angiogenic factors.

Salinomycin
Isolated in Japan in 1974 from Streptomyces albus, salinomycin represents a carboxylic polyether ionophore antibiotic, leading to disturbances in Na + /K + ion equilibrium in biological membranes and, due to reduced K + concentration, in mitochondria and cytoplasm, resulting in cell apoptosis. 15,16Salinomycin is also active through several other mechanisms in tumors of various locations.It inhibits the conserved Wnt pathway, providing CSCs with the ability to self-replace and resist radiotherapy.Salinomycin also reduces the resistance of CSCs to chemotherapy as it affects the activity of ABC transporters.][19] Studies by Zhang et al. in in vivo and in vitro conditions demonstrated that salinomycin induces apoptosis in the cells of ovarian cancer resistant to cisplatin, apoptosis linked to the pathway of mitogen-activated protein kinase (MAPK). 20Studies by Kaplan and Teksen on the stable ovarian cancer cell line (OVCAR-3) demonstrated the activity of salinomycin: 40% of cells underwent apoptosis within 24 h of exposure. 21A decrease was noted in the expression of the anti-apoptotic gene Bcl2.
Chung et al., using the OVCAR-3 ovarian cancer cell line, isolated stem cells of markers CD44 + and CD117 + , linked to chemoresistance. 22They used paclitaxel + salinomycin.Supplementation with salinomycin significantly inhibited the growth of the cells.Another mechanism of salinomycin activity is linked to the activity of death receptor 5 (DR5) and the activation of apoptosis-initiating caspase 8. Binding of DR5 to its ligand leads to alterations in death domain (DD, part of the caspase), mobilizing the enzymatic cascade which results in the death of the cell. 23nother mechanism of apoptosis in the ovarian cancer cells resistant to cisplatin involves the inhibitory effect of salinomycin on the nuclear transcription factor NF-kB, which affects the control of several other genes. 24n practice, salinomycin is also being used by prof.Cord Naujokat in Heidelberg, Germany, obtaining good results among invasive carcinoma in screening studies.The administration of salinomycin has resulted in the inhibition of disease progress over an extended period of time.It is worth noting that acute side effects were rare and serious long-term adverse side effects were not observed. 17t has also been demonstrated that salinomycin is able to kill CSCs, regular tumor cells and highly indolent tumor cells displaying resistance to cytotoxic drugs, radiation and the induction of apoptosis. 16p to now, the results obtained from in vivo studies on human xenograft mice and also clinical studies have revealed that salinomycin is able to effectively eliminate CSCs and to induce partial clinical regression in therapyresistant cancers. 17

Metformin
Metformin represents a drug of the biguanide group, used orally in patients with diabetes type II, particularly in overweight/obese patients.Population studies have proved that metformin-treated patients manifested a lowered risk of malignant tumors in several locations, including the preventive effect of the drug in ovarian cancer.In comparison to patients not using metformin, women who develop ovarian cancer and being treated with this drug are older and diagnosed more frequently at earlier International Federation of Gynecology and Obstetrics (FIGO) clinical stages.][31] Studies on the SKOV3 and A2780 cell lines of ovarian cancers conducted by Zhang et al. demonstrated that low doses of metformin selectively inhibit CSCs with markers CD44+ and CD117+, and inhibit epithelial mesenchymal transition (EMT). 32Epithelial mesenchymal transition is linked to the activity of transcription factors and the activation of several genes, resulting in the inhibition of apoptosis and an increased mobility of cells, linked to metastases.Shank et al. confirmed better prognosis in ovarian cancer patients treated with metformin. 29The authors expressed the opinion that the mechanism of its action involved a reduction in the CSC population with ALDH1+ marker, reduced cell proliferation and inhibited angiogenesis.They argued that the results provided rationale for the application of metformin in patients treated due to ovarian cancer.

Clostridium perfringens enterotoxin
Natural receptors for Clostridium perfringens enterotoxin (CPE) involve claudins 3 and 4. Claudins 3 and 4 represent the family of tight junction proteins responsible for the integrity of the cell membrane and for its functions.In ovarian cancer, overexpression of claudins 3 and 4 is noted; they play a critical role in invasion and metastases as well as in resistance to chemotherapy.They may provide an attractive target in the treatment of cancer resistant to platins. 33asagrande et al. described the mechanism through which CPE becomes activated in ovarian cancer. 34They examined a small number of ovarian CSCs in vitro and in vivo, carrying the marker of CD44+.They exhibited a high expression of claudin 4. Following the exposure to CPE, the cells underwent apoptosis.Following a sublethal dose of CPE, ovarian cancer xenografts to mice regressed, resulting in a long survival of the animals.Similar results were described by other authors conducting research on ovarian CSCs, CD44+, resistant to paclitaxel and carboplatin.Clostridium perfringens enterotoxin may represent an unconventional therapy eradicating ovarian cancer CSCs. 35w about an anti-angiogenic therapy?
Bevacizumab, an anti-angiogenic humanized monoclonal antibody, is used in first line therapy in advanced ovarian cancer.According to Rauh-Hain et al., relapses of ovarian cancer in patients treated with bevacizumab were located in other body parts than in patients not treated with the antibody. 36Those receiving bevacizumab more frequently developed relapses in the pleura, lungs, brain, liver, and lymph nodes than patients on the anti-angiogenic treatment.
Since relapses, as presented above, were linked to CSC metastases, one can speculate on a link between anti-angiogenic therapy and CSC activity.Kaplan et al. found that CSCs with expression of VEGFR1+ promote chemotaxis and adherence of circulating tumor cells. 37VEGFR1+ cells establish specific "premetastatic" niches, forming cellular clusters there before the inflow of tumor cells.The application of the antibody specific to VEGFR1+ or the removal of the cells prevents metastases.In addition, VEGFR1+ cells manifest expression of VLA4 (known as integrin α 4β1 ), the ligand of which involves fibronectin taking part in cell interaction with the matrix.According to Chau et al., the identification of factors targeted at CSCs and associating them with angiogenesis inhibitors might warrant their better clinical efficacy. 38The authors also suggest that antiangiogenic therapy leads to tumor hypoxia, resulting in the growth of the CSC population.In their study, Conley et al. presented a similar hypothesis. 39The problem of anti-angiogenic therapy in ovarian cancer and, in particular, its relationship with CSCs, is extremely intriguing.Anti-angiogenic action based on immunotherapy (vaccine) might uncover another relationship with CSCs. 40Regarding future directions in the treatment of ovarian cancer, it would seem that combined therapy -cytostatics, anti-angiogenics and anti-CSCs -is a very promising option.