Survival pathways in CSCs

Resistance of cancer cells to CTL-mediated immunotherapyResistance to targeted anti-cancer therapeutics. Benjamin Bonavida, Salem Chouaib (Eds). Springer International Publishing Switzerland (2015)

The signaling pathways, which regulate normal stem cell self-renewal, lead to tumorigenesis when dysregulated; a comprehensive understanding of the pathways involved in development, “stemness” and apoptosis, is considered to be a very important goal in cancer therapy. The most important signaling pathways that regulate normal and cancer stem cell functions are: Notch, Wnt, BMP and Sonic-Hedgehog.

The Notch signaling pathway is evolutionarily conserved and has profound, context-dependent phenotypic consequences because it is involved in the maintenance of stem cells and in differentiation regulation. In both humans and rodents, the Notch genes encode four distinct members (from Notch1 to Notch4) of a transmembrane heterodimeric receptor family. In physiologic conditions, Notch ligands (Delta and Jagged) binding induces the Notch receptor intracellular domain (Notch-IC) release via a cascade of proteolytic cleavages catalyzed by a disintegrin and metalloprotease (ADAM) and γ-secretase (GS) proteases. Notch-IC translocates into the nucleus and modulates the gene expression by binding the transcription factor, CBF1/Su(H)/Lag-1 (CSL), and recruiting co-activators, such as recombining binding protein suppressor of hairless (R) and mastermind-like protein 1 (M) [65] (Fig. 1.1a). The aberrant activation of this pathway contributes to tumorigenesis [66–70]. With the notable exception of epidermal keratinocytes where Notch-1 functions as a tumor suppressor [71], the inappropriate activation of the Notch pathway results in the stimulation of proliferation, restriction of differentiation and prevention of apoptosis in T-cell acute lymphoblastic leukemia [69], breast cancer [72, 73], melanoma [74], lung adenocarcinoma [75] and others. Therefore, a possible anticancer therapy goal may be the Notch signaling inhibition that is achieved at many different levels. It is possible to interfere with receptor activation by blocking ligand-induced conformational changes [76] and releasing the Notch-IC receptor by blocking the ADAM [77] or GS proteases cleavage [53, 65, 78]. In addition, Notch signaling could be inhibited by disrupting protein–protein interactions involved in nuclear events, including the assembly of co-activators [79, 80]. The γ-secretase inhibitors (GSIs) and monoclonal antibodies (mAbs), which block Notch receptors, are currently in the beginning stages of clinical trials [81, 82]. Moreover, mAbs that target Notch ligand Delta-like 4 have been shown to inhibit Notch signaling in endothelial cells by inducing disorganized angiogenesis [83]. In the platinum-resistant ovarian cancer, the inhibition of Notch signaling by a GSI and conventional Paclitaxel chemotherapy, synergistically reduced xenograft growth [84]. In intestinal crypts, where the staminal cell niche is located, Notch directs proliferation when Wnt signaling activity is high and promotes enterocyte differentiation when Wnt activity levels are reduced [85].

 Resistance of Cancer Cells to CTL-Mediated Immunotherapy-Springer International Publishing (2015) 1.1

Fig. 1.1. Pathways involved in CSC survival and differentiation. (a) Notch signaling. Notch signaling relies on the activation of Notch receptors by Delta and Jagged ligands expressed in a neighbor cell. The release of Notch-IC, subsequent to the two proteolytic events catalyzed by ADAM and GS proteases, leads to the transcription of target genes by binding the transcription factor CSL and recruiting the co-activators R and M. (b) Canonical Wnt/β-catenin signaling. Wnt binds to FZ, which recruits LRP5/6 as co-factor and interacts with Dsh. β-catenin cytoplasmatic localization is regulated by a destruction complex formed by APC, Axin2, GSK3βCK1, which directs its degradation by ubiquitination. In presence of Wnt ligands, Dsh inhibits GSK3 and the destruction complex disassembles allowing β-catenin to shift to the nucleus. (c) BMP signaling. The heterodimerization of BMPR receptors induced by BMP proteins promotes the phosphorylation of SMAD 1,5,8 and their association with SMAD 4. The complex formed enters into the nucleus and stimulates the target genes’ transcription aided by Runx2 and a cofactor (C). (d) Hedgehog signaling. Signaling by Hh depends on the interaction between the membrane proteins SMO and PTCH. When bound to Hh, PTCH does not repress SMO, which in turn activates GLI transcription factors

Wnt proteins constitute a family of signaling molecules that regulate cell-to-cell interactions during development. They are secreted glycoproteins that bind to the extra-cellular domain of the Frizzled (FZ) receptor, a seven-transmembrane protein that requires different co-receptors to mediate three different Wnt pathways:

(1) the canonical Wnt/β-catenin cascade;

(2) the non canonical planar cell polarity (PCP) pathway; and (3) the Wnt/Ca2+ pathway.

In the canonical Wnt pathway, the co-factor low-density-lipoprotein-related protein5/6 (LRP5/6) interacts with the cytoplasmatic phosphoprotein Dishevelled (Dsh) [86]. This interaction causes an accumulation of β-catenin in the cytoplasm and its translocation into the nucleus, where it attracts, as co-activator, some transcription factors belonging to the T-cell factor-1 and lymphoid enhancing factor-1 TCF-1/LEF-1 family as well as regulating gene transduction. In the absence of Wnt ligands, a destruction complex formed by Axin2, adenomatosis polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), degrades β-catenin by targeting for ubiquitination. The canonical Wnt pathway activation produces the translocation of the negative Wnt regulator, Axin2, to the plasma membrane where it binds to the cytoplasmatic tail of LRP-5/6. Thus, Axin2 becomes de-phosphorylated and its stability is decreased. Moreover, Dsh inhibits the GSK3 activity of the destruction complex allowing the β-catenin accumulation in the nucleus (Fig. 1.1b) [85].

The Wnt canonical signaling is important in many developmental processes and in the regulation of self-renewal in normal and CSCs. In particular, the Wnt target gene leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) marks stem cells in multiple adult tissues and cancers [87]. A germline APC mutation is the genetic cause of a hereditary colorectal cancer syndrome called Familiar Adenomatous Polyposis (FAP) [88, 89]. The cytoplasmatic interaction of APC with β-catenin provided the first connection between the Wnt pathway and human cancer [90, 91]. In intestinal epithelial cells in which APC is mutated, the constitutive β-catenin/TCF4 complex activates a genetic program within the crypt of stem/progenitor cells for the maintenance of cell proliferation [92]. In rare cases of colorectal cancer where APC is not mutated, Axin2 or β-catenin could be mutant [93, 94]. Loss-of-function mutations in Axin2 have been found in hepatocellular carcinomas whereas, oncogenic β-catenin mutations occur in a wide variety of solid tumors [95]. Concerning the colon cancer crypt, β-catenin induces the expression of EphB receptors which, interact with ephrin ligands inducing cells proliferation and thereby tumor progression [96, 97].

The non-canonical PCP pathway is one of the two Wnt pathways that does not involve β-catenin. After binding to Fz and its co-receptor (NRH1, Ryk, PTK7, or ROR2), the Wnt4, Wnt5a and Wnt11 ligands promote the pathway activation. These receptors form a complex inclusive of Dsh and Dishevelled-associated activator of morphogenesis 1 (DAAM1), which activate the small G-protein Rho and the Rho-associated kinase (ROCK), one of the cytoskeleton major regulators. The noncanonical Wnt pathway was shown to regulate both cell polarity and movements of dorsal mesodermal cells during neural tube closure [98]. ROCK activation has also been implicated in the cancer stem cells cytoskeleton organization and thereby in their migration and metastasis formation [99, 100].

The non-canonical Wnt signaling is reported to antagonize β-catenin-dependent transcription, suggesting an important anti-oncogenic effect [101]. However, a core PCP pathway scaffolding protein VANGL1 has been shown to promote metastasis in colon cancer. Moreover, it has been demonstrated that Wnt5a promotes mammosphere formation via a non-canonical mechanism which involves ROR2 as co-receptor [102].

The Wnt/Ca2+ pathway shares many components of the PCP pathway, but plays a different role in stimulating intracellular Ca2+ release by ER [103, 104]. The intracellular calcium accumulation activates several Ca2+ sensitive proteins, including protein kinase C [105] and calcium/calmodulin-dependent kinase II [106]. In melanomas, the activation of PKC as a result of the Wnt/Ca2+ pathway is involved in cell proliferation and metastasis. Thus, targeting this pathway could be relevant to cancer therapy [107].

The Bone morphogenetic protein 4 (BMP4) is able to activate a differentiation program and stimulate apoptosis in colon cancer stem cells. This reduces the β-catenin activation through inhibition of the PI3K/AKT pathway and up-regulation of the Wnt-negative modulators [108].

Bone Morphogenetic Proteins (BMPs) are secreted signaling molecules that comprise a subfamily of the TGF-β family. There are at least 20 structurally and functionally related BMPs, most of which play a role in embryogenesis and morphogenesis in various tissues and organs. BMP signaling depends on the heterodimerization of type I and II BMP receptors (BMPR) that lead to phosphorylation of the downstream molecules SMAD 1,5,8 and their association with SMAD 4. This complex translocates into the nucleus where it interacts with Runt-related transcription factor 2 (Runx2) and a cofactor (C) (Fig. 1.1c) [85]. Considering that BMP4 prevents cell proliferation and stimulates apoptosis by inducing the BAX expression and downregulating BCL-2 and Bcl-xL levels, the BMPR agonist could be useful in targeting cancer stem cells by activating a differentiation program and so potentiating the chemotherapy’s cytotoxicity through BCL-2 negative inhibition in glioblastoma (GBM) [109]. It has recently been demonstrated that the BMP7 variant is a possible and innovative approach to the treatment of GBM since it decreases tumor growth in orthotopic mice models and stem cell markers expression, while enhancing differentiation markers expression [110].

The Hedgehog (Hh) signaling pathway plays very important functions in growth regulation, survival and fate during embryonic development and in the maintenance/ repair of adult tissues. The Hh proteins initiate signaling by binding to the receptor Patched (PTCH). The subsequent receptor internalization alleviates the inhibitory effect of Patched on the 7-transmembrane protein Smoothened (SMO), which in turn activates the Hh pathway. Thus, the derepressed SMO activates GLI transcription factors, which translocate directly to the nucleus and drive the transcription of target genes (Fig. 1.1d) [111]. Deregulation of this pathway has been associated with tumorigenesis or tumor growth acceleration in a wide variety of tissues. Basal cell carcinoma, medulloblastoma, gastric cancer and pancreatic cancer bear mutations in components of the Hh pathway [112–116]. Moreover, the Hh pathway plays important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells [117]. In GBM the treatment with an Hh pathway inhibitor, cyclopamine, caused a 40–60 % reduction in tumor growth and of the tumorigenic potential of CSCs [118].

Finally, it is demonstrated that, in the human pancreatic adenocarcinoma cell line, inhibiting the Hh pathway by cyclopamine, depressed tumor spheres self-renewal [119] and reversed gemcitabine resistance [120].

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