Cancer immunology. Bench to bedside immunotherapy of cancers. Nima Rezaei (Ed.). Springer-Verlag (2015)
Cancer stem cells account for a minor fraction of a tumor population; nevertheless, they could play a central role in treatment failure and relapse. At present, neoadjuvant treatment targets the proliferative potential of the tumors by killing rapidly dividing cells within the bulk of the tumor. However, even in the event of a considerable shrinkage of the tumor burden following a highly effective therapy that successfully affects the vast majority of tumor cells, CSCs could be unaffected. In glioblastoma, CD133+ CSC population was found to be enriched after radiation and exhibited lower rates of apoptosis in response to chemotherapy  in comparison with CD133-. In breast cancer, significantly increased levels of cells expressing CSC markers have been reported in residual tumor cell populations of patients after conventional chemotherapy [260, 261]. Moreover, in pancreatic cancer, CD133+ cells showed increased resistance to chemotherapeutic agents . Indeed, CSCs display enhanced resistance to conventional cytotoxic agents (i.e., chemotherapy and ionizing radiation, inducing cell death mostly by DNA damage) due to numerous strategies: quiescence propensity, enhanced DNA repair, upregulated cell cycle control mechanisms, free-radical scavenging mechanisms, and specific interaction with stromal microenvironment. Development of targeted therapies based on inhibition of these features could allow to overwhelm treatment resistance, in order to eradicate CSCs and achieve long-lasting tumor remission.
Enhanced DNA repair
Cancer cells improperly activate DNA repair pathways, implied in preservation of genome integrity, in order to overcome standard anticancer treatments. DNA repair pathways include :
- Nucleotide excision repair (NER) that corrects massive helix-distorting lesions
- Base excision repair (BER) that targets point base modifications
- Mismatch repair (MMR), removing mispaired nucleotides in the event of replication errors
- Monoenzymatic direct repair, involving O6-methylguanine methyltransferase (MGMT), that performs a one-step methyl transfer reaction
- Double-strand break (DSB) recombinant repair, a complex and cycle-dependent mechanism that encompasses homologous recombination repair (HRR), prevalent in cycling phases, and nonhomologous end-joining (NHEJ) that is eminent in G1 phase. This pathway can consequently lead to activation of secondary effectors, such as ataxia telangiectasia mutated (ATM), ataxia telangiectasia/ Rad3-related kinase (ATR), and checkpoint kinases (Chk1 and Chk2)
The connection between DNA repair signals and CSC chemoresistance has been highlighted in glioma cell lines, showing that enhanced activation of ATM and Chk1 in CD133+ resulted in increased survival after irradiation, while radiosensitivity was restored with pharmacological inhibition . Enhanced DNA repair ability has been also observed in breast CSCs . Early clinical trials with DNA repair inhibitors, such as the MGMTdepleting agents O6-benzylguanine, in association with chemotherapy showed disappointing results [264, 265]. Attention has been drawn on PARP inhibitors, in reason of PARP-1 and PARP-2 involvement in single-strand repair via the BER pathway: these molecules have been particularly tested in breast cancers harboring BRCA mutation that relies on BER due to impaired HRR [266, 267].
A phase II multicenter study conducted in patients with advanced, refractory, BRCA-mutation carrier breast cancer evaluated two different schedules of the oral PARP inhibitor olaparib (AZD2281) in two sequential cohorts: overall response rates were 41% (11 patients) and 22% (6 patients) with olaparib at 400 and 100 mg, respectively, with an acceptable toxicity . Iniparib (BSI-201) has been tested in a randomized, phase II study trial in association with carboplatin/gemcitabine doublet for treating metastatic triple negative breast cancers, showing a significant clinical benefit in the experimental arm . However, the subsequent phase III trial failed to confirm any Efficacy . Chk1 inhibitors (AZD7762, PF-477736, SCH900776, LY2606368) are undergoing early phases of clinical development.
Research on the contribution of polycomb group proteins, such as BM1, to the DNA damage response pathways is another promising area of investigation .
Hypoxia is involved in radioresistance, as cells located in areas of low-oxygen tensions are less exposed to reactive oxygen species (ROS)mediated damage . It has been reported that lower levels of oxidative radicals are detected after irradiation in CSC-enriched MCF-7 breast cancer cell mammospheres in comparison with monolayer cultures , which could be explained by improved free-radical scavenging pathways. For example, overexpression of glutathione-related genes (Gclm and Gss) has been observed in CSCs: selective inhibition by buthionine sulfoximine (BSO), a glutamatecysteine ligase inhibitor, induced a decrease in the colony-forming ability and restored radiation sensitivity in CSC models . As previously stated, also HIF expression, in its two isoforms HIF1a and HIF2a, is implicated in CSC promotion and maintenance . In some hematological malignancies, CSCs are sustained by high levels of HIF1a (under normoxia) that promotes gene expression of the stem cell transcription factor Hes1, via the stimulation of the Notch pathway. Echinomycin, an HIF1a inhibitor, was selectively effective on these CSCs . Recent experiences by Lee et al. suggest that sorafenib, in combination with radiotherapy, could enhance the efficacy of irradiation on CSCs, by inhibition of HIF-1a in an in vitro breast cancer model .
Experimental evidences indicate that, both in vitro and in vivo, subpopulations of slow-cycling tumor cells are mostly spared by DNA-damage-induced death as compared to the bulk of tumor cells . It is known that cells change in their sensitivity to DNA-damaging agents all along the division cycle, ranging from extreme sensitivity in the mitotic phase and increased resistance in late S-phase . Specific therapies might induce CSCs to differentiate into more mature tumor cells, thus limiting their tumorigenic and invasive potential. Salinomycin has been described as the first “quiescence-disrupting” compound that is able to decrease the proportion of CSC phenotypic breast cancer cells and to selectively eradicate the tumor, by inducing terminal epithelial differentiation . It has been also suggested that histone deacetylase (HDAC) causes a lysin residues epigenetic modification and is responsible of chromatin condensation of CSCs . A novel class of therapeutic agents, the epigeneticacting histone deacetylase inhibitors (HDACi), is currently under investigation in this setting .
Notch-targeting agents like gamma secretase inhibitors (GSIs), a category of compounds blocking the release of the Notch intracellular domain, showed promising activity in preclinical studies and are currently undergoing clinical evaluation . RO4929097, a new Notch inhibitor , has been extensively studied for toxicity in a number of settings, both as a single agent  and in combination with standard chemotherapy ; phase II trials are ongoing for patients with recurrent or progressive glioblastoma (http://clinicaltrials.gov: NCT01122901); nevertheless, disappointing results were observed when used as a single agent in metastatic colorectal cancer . Another approach exploiting antibodies against delta-like 4 ligand (DLL4), a component of Notch signaling pathway, achieved inhibition of the expression of Notch target genes and reduced proliferation of tumor cells in a mouse model of human colon cancer, either alone or in combination with irinotecan .
- Hedgehog signaling could be pharmacologically inhibited by targeting Smo. Preclinical studies with pancreatic cancer models showed that pharmacological Smo inhibition was effective against CD133+ pancreatic CSCs, with enhanced apoptosis, probably associated with Fas and death receptor (DR) overexpression , while combined treatment with Sonic Hedgehog and mTOR inhibitors, together with standard chemotherapy, has proved to be capable of eliminating pancreatic CSCs in in vitro and in vivo models . Preliminary experience with Smo antagonist GDC-0449 (vismodegib) showed significant Efficacy, with mild toxicity, in patients affected by basal cell carcinoma, leading to FDA approval in this setting on January 2012 ; nevertheless, clinical activity in other solid tumors was controversial  and rapid onset of Smo acquired mutation was observed in one patient treated for medulloblastoma that progressed after initial stabilization of disease . Other strategies to achieve Hedgehog inhibition encompass Gli antagonists  or, indirectly, modulation of other signaling pathways, like EGFR and TGFβ [294, 295].
- WNT: antibodies directed toward Wnt receptor Frizzled7 (FZD7) reduce clonogenicity and tumorigenicity in preclinical models of Wilms’ tumor; a synergistic gain in Efficacy could be obtained with the addition of the analogues of Dickkopf1 (Dkk1), a secretase related to the differentiation of CD44+CD24 low breast CSCs  that prevents the formation of the Frizzled-Wnt-LRP6 complex . Another strategy could imply direct inhibition of LRP6 or FZD7, which proved effective in suppression of tumor growth in in vitro models of triple negative breast cancer . Kendizorra et al. described enhanced radioresistance in rectal cancer cell lines overexpressing Wnt transcription factor T cell factor (TCF-4), while sensitivity was restored by silencing TCF-4 . RO4929097, an investigational Wnt pathway inhibitor, is currently being evaluated in a phase I trial recruiting breast cancer patients (http://clinicaltrials. gov: NCT01351103). Further advances in the knowledge of the Hippo pathway that intersects both the Wnt and Notch pathways could allow the development of new generation targeted therapies .
- mTOR/AKT: inhibition of the PI3K/AKT/ mTOR signaling pathway could be effective in restoring sensitivity to chemotherapy and radiation in CSCs that aberrantly activate this pathway . Strategies to achieve this goal encompass pharmacological abrogation of AKT, mTOR inhibitors, and PI3K antagonists. Novel AKT inhibitors have shown to be promising in in vitro/in vivo antitumor activity, in combination with chemotherapeutic agents , and are currently undergoing phase I/II trials. mTOR inhibitors have been extensively studied in association with standard chemotherapy, but their activity on CSCs has not been specifically investigated yet. On stem cells from HER2-overexpressing primary breast cancer cells and on BT474 breast cancer cell line, it has been recently highlighted that everolimus, in combination with trastuzumab, provides a rationale for strategies that overcome resistance to HER2-directed agents .