Overexpression of Multifunctional Protein p32 Promotes a Malignant Phenotype in Colorectal Cancer Cells.

p32 is a multifunctional and multicompartmental protein that has been found upregulated in numerous adenocarcinomas, including colorectal malignancy. High levels of p32 expression have been correlated with poor prognosis in colorectal cancer. However, the functions performed by p32 in colorectal cancer have not been characterized. Here we show that p32 is overexpressed in colorectal cancer cell lines compared to non-malignant colon cells. Colon cancer cells also display higher nuclear levels of p32 than nuclear levels found in non-malignant cells. Moreover, we demonstrate that p32 regulates the expression levels of genes tightly related to malignant phenotypes such as HAS-2 and PDCD4. Remarkably, we demonstrate that knockdown of p32 negatively affects Akt/mTOR signaling activation, inhibits the migration ability of colon malignant cells, and sensitizes them to cell death induced by oxidative stress and chemotherapeutic agents, but not to cell death induced by nutritional stress. In addition, knockdown of p32 significantly decreased clonogenic capacity and in vivo tumorigenesis in a xenograft mice model. Altogether, our results demonstrate that p32 is an important promoter of malignant phenotype in colorectal cancer cells, suggesting that it could be used as a therapeutic target in colorectal cancer treatment.

Signaling Pathways Involved in Nutrient Sensing Control in Cancer Stem Cells: An Overview.

Cancer cells characteristically have a high proliferation rate. Because tumor growth depends on energy-consuming anabolic processes, including biosynthesis of protein, lipid, and nucleotides, many tumor-associated conditions, including intermittent oxygen deficiency due to insufficient vascularization, oxidative stress, and nutrient deprivation, results from fast growth. To cope with these environmental stressors, cancer cells, including cancer stem cells, must adapt their metabolism to maintain cellular homeostasis. It is well- known that cancer stem cells (CSC) reprogram their metabolism to adapt to live in hypoxic niches. They usually change from oxidative phosphorylation to increased aerobic glycolysis even in the presence of oxygen. However, as opposed to most differentiated cancer cells relying on glycolysis, CSCs can be highly glycolytic or oxidative phosphorylation-dependent, displaying high metabolic plasticity. Although the influence of the metabolic and nutrient-sensing pathways on the maintenance of stemness has been recognized, the molecular mechanisms that link these pathways to stemness are not well known. Here in this review, we describe the most relevant signaling pathways involved in nutrient sensing and cancer cell survival. Among them, Adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway, mTOR pathway, and Hexosamine Biosynthetic Pathway (HBP) are critical sensors of cellular energy and nutrient status in cancer cells and interact in complex and dynamic ways.

Canonical and non-canonical Wnt signaling are simultaneously activated by Wnts in colon cancer cells.

The Wnt signaling pathway is a crucial regulator of the intestinal epithelium homeostasis and is altered in most colon cancers. While the role of aberrant canonical, ß-catenin-dependent Wnt signaling has been well established in colon cancer promotion, much less is known about the role played by noncanonical, ß-catenin-independent Wnt signaling in this type of cancer. This work aimed to characterize the noncanonical signal transduction pathway in colon cancer cells. To this end, we used the prototype noncanonical ligand, Wnt5a, in comparison with Wnt3a, the prototype of a canonical ß-catenin activating ligand. The analysis of the expression profile of Wnt receptors in colon cancer cell lines showed a clear increase in both level expression and variety of Frizzled receptor types expressed in colon cancer cells compared with non-malignant cells. We found that Wnt5a activates a typical Wnt/Ca++ - noncanonical signaling pathway in colon malignant cells, inducing the hyperphosphorylation of Dvl1, Dvl2 and Dvl3, promoting Ca++ mobilization as a result of phospholipase C (PLC) activation via pertussis toxin-sensitive G-protein, and inducing PLC-dependent cell migration. We also found that while the co-receptor Ror2 tyrosine kinase activity is not required for Ca++ mobilization-induced by Wnt5a, it is required for the inhibitory effects of Wnt5a on the ß-catenin-dependent transcriptional activity. Unexpectedly, we found that although the prototype canonical Wnt3a ligand was unique in stimulating the ß-catenin-dependent transcriptional activity, it also simultaneously activated PLC, promoted Ca++ mobilization, and induced Rho kinase and PLC-dependent cell migration. Our data indicate, therefore, that a Wnt ligand can activate at the same time the so-called Wnt canonical and noncanonical pathways inducing the formation of complex signaling networks to integrate both pathways in colon cancer cells.

Functional Interaction of Hypoxia-Inducible Factor 2-Alpha and Autophagy Mediates Drug Resistance in Colon Cancer Cells.

Hypoxia and the accumulation of hypoxia-inducible factors (HIFs) in tumors have been associated with therapeutic resistance and with autophagy establishment. We examined the effects of stable knockdown of HIF-1α or HIF-2α expression on autophagy and drug resistance in colon cancer cells. We found that under normoxic conditions, malignant cells exhibit increased basal levels of autophagy, compared with non-malignant cells, in addition to the previously reported coexpression of HIF-1α and HIF-2α. Knockdown of HIF-1α or HIF-2α expression resulted in increased autophagic and apoptotic cell death, indicating that the survival of cells is HIF-dependent. Cytotoxic-induced cell death was significantly increased by knockdown of HIFs but not by autophagy inhibition. Strikingly, although malignancy-resistant cells were sensitized to death by nutrient stress, the combination with HIF-2α depletion, but not with HIF-1α depletion, induced severe cell death. Oxidative stress levels were significantly increased as a result of HIF-2α specific inhibition or silencing suggesting that this may contribute to sensitize cells to death. The in vitro results were confirmed in vivo using a xenograft mouse model. We found that coordinated autophagy and mTOR inhibition enhanced cell death and induced tumor remission only in HIF-2α-silenced cells. Finally, using a specific HIF-2α inhibitor alone or in combination with drugs in patient-derived primary colon cancer cells, overcame their resistance to 5-FU or CCI-779, thus emphasizing the crucial role played by HIF-2α in promoting resistance and cell survival.

O-GlcNAcylation Is Involved in the Regulation of Stem Cell Markers Expression in Colon Cancer Cells.

The dynamic O-linked-N-acetylglucosamine posttranslational modification of nucleocytoplasmic proteins has emerged as a key regulator of diverse cellular processes including several hallmarks of cancer. However, the role played by this modification in the establishment of CSC phenotype has been poorly studied so far and remains unclear. In this study we confirmed the previous reports showing that colon cancer cells exhibit higher O-GlcNAc basal levels than non-malignant cells, and investigated the role played by O-GlcNAcylation in the regulation of CSC phenotype. We found that the modification of O-GlcNAcylation levels by pharmacological inhibition of the O-GlcNAc-transferase enzyme that adds O-GlcNAc (OGT), but not of the enzyme that removes it (OGA), increased the expression of all stem cell markers tested in our colon malignant cell lines, and induced the appearance of a double positive (CD44+/CD133+) small stem cell-like subpopulation (which corresponded to 1-10%) that displayed very aggressive malignant phenotype such as increased clonogenicity and spheroid formation abilities in 3D culture. We reasoned that OGT inhibition would mimic in the tumor the presence of severe nutritional stress, and indeed, we demonstrated that nutritional stress reproduced in colon cancer cells the effects obtained with OGT inhibition. Thus, our data strongly suggests that stemness is regulated by HBP/O-GlcNAcylation nutrient sensing pathway, and that O-GlcNAc nutrient sensor represents an important survival mechanism in cancer cells under nutritional stressful conditions.

Dishevelled stability is positively regulated by PKCζ-mediated phosphorylation induced by Wnt agonists.

Dishevelled (Dvl) proteins are central mediators of both canonical and non-canonical Wnt signaling. It is well known that, upon Wnt stimulation, Dvl becomes phosphorylated. However, how Wnt-induced phosphorylation of Dvl is regulated and its consequences are poorly understood. Here we found that Dvl proteins are overexpressed in colon cancer cells. In addition, we found that Wnt3a treatment rapidly induces hyperphosphorylation and stabilization of Dvl2 and Dvl3. The latter can be blocked by inhibition of Protein Kinase C (PKC)α, PKCδ, and PKCζ isoforms. We also found that Wnt3a-induced phosphorylation of Dvl3 by PKCζ is required to avoid Dvl3 degradation via proteasome. This demonstrated, to our knowledge for the first time, that hyperphosphorylation of Dvl by PKCζ results in Dvl stabilization. This is clear contrast with the consequences reported to date of CK1δ/ε-mediated Dvl phosphorylation upon Wnt treatment. Mapping the interaction domain between PKCζ and Dvl3 indicated that, although the Dvl-DIX domain is required to stabilize PKCζ-phosphorylated Dvl, it is not the region phosphorylated by this kinase. Our data show that the Dvl-DEP domain, required for specific interaction with PKCζ, is the site phosphorylated by this kinase, and also probably the Dvl-C terminus. Our findings suggest a model of positive regulation of PKCζ-mediated Dvl signaling activity, to produce a strong and sustained response to Wnt3a treatment by stabilizing Dvl protein levels.

Glycogen Synthase Kinase 3ß Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists.

The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3ß (GSK-3ß) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3ß interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3ß redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3ß at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3ß in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3ß. In vitro activity assays demonstrated that GSK-3ß phosphorylation mediated by PKCζ enhanced GSK-3ß activity. We mapped Ser147 of GSK-3ß as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3ß activity, resulting in ß-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3ß basal activity. Thus, we found that PKCζ phosphorylates GSK-3ß at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3ß activity in opposite manners in normal and malignant colon cells.

Protein phosphatase 2A is essential to maintain active Wnt signaling and its Aß tumor suppressor subunit is not expressed in colon cancer cells.

Canonical Wnt signaling is altered in most cases of colorectal cancer. Experimental evidence indicates that protein phosphatase 2A (PP2A) may play either positive or negative roles in Wnt signaling but its precise in vivo functions remain elusive. In this work, using colon cultured cell lines we showed that basal PP2A activity is markedly reduced in malignant cells compared to non-malignant counterparts. We found that whereas normal or cancer cells displaying not altered Wnt signaling express mRNAs coding for PP2A-A scaffold α and ß isoforms, cancer cells which have altered Wnt signaling do not express the Aß isoform mRNA. Remarkably, we found that the Aß protein levels are lost in all colon cancer cells, and in patients' tumor biopsies. In addition, all cancer cells exhibit higher levels of RalA activity, compared to non-malignant cells. Rescue experiments to restore Aß expression in malignant RKO cells, diminished the RalGTPase activation and cell proliferation, indicating that the Aß isoform acts as tumor suppressor in colon cancer cells. Reciprocal co-immunoprecipitation and immunofluorescence studies showed that the PP2A-C and -Aα subunits, expressed in all colon cells, interact in vivo with ß-catenin only in malignant cells. Selective inhibition of PP2A did not significantly affect cellular apoptosis but induced dose-dependent negative effects in ß-catenin-mediated transcriptional activity and in cell proliferation of malignant cells, indicating that the residual PP2A activity found in malignant cells, mediated by -C and Aα core subunits, is essential to maintain active Wnt signaling and cell proliferation in colon cancer cells.

Hypoxia-inducible factors modulate the stemness and malignancy of colon cancer cells by playing opposite roles in canonical Wnt signaling.

This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling. Under normoxia, we determined that both HIF-1α and HIF-2α are expressed in human colon cancer cells but not in their non-malignant counterparts. The stable knockdown of HIF-1α or HIF-2α expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1α depletion exerting greater effects. Knockdown of these two HIF transcripts induced different and even opposite effects on ß-catenin transcriptional activity in colon cancer cells with different genetic Wnt signaling pathways. In SW480 cells, HIF-2α knockdown did not affect ß-catenin levels, increasing the transcriptional activity of ß-catenin by inducing its nuclear accumulation, whereas HIF-1α silencing negatively affected the stability and transcriptional activity of ß-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin. In addition, although HIF-1α depletion induced a reversal of the epithelial-to-mesenchymal transition (EMT), HIF-2α silencing altered the expression of the stem cell markers CD44, Oct4, and CD24 and of the differentiation marker CK20 in the opposite direction as HIF-1α silencing. Remarkably, HIF-2α knockdown also enhanced ß-catenin transcriptional activity under hypoxia in cells that displayed normal Wnt signaling, suggesting that the gene negatively modulates canonical Wnt signaling in colon cancer cells. Taken together, our results indicate that HIFs play opposing roles in canonical Wnt signaling and are essential for the stemness and malignancy maintenance of colon cancer cells.

Protein kinase C delta negatively modulates canonical Wnt pathway and cell proliferation in colon tumor cell lines.

The tumor suppressor Adenomatous Polyposis coli (APC) gene is mutated or lost in most colon cancers. Alterations in Protein kinase C (PKC) isozyme expression and aberrant regulation also comprise early events in intestinal carcinomas. Here we show that PKCδ expression levels are decreased in colon tumor cell lines with respect to non-malignant cells. Reciprocal co-immunoprecipitation and immunofluorescence studies revealed that PKCδ interacts specifically with both full-length (from non-malignant cells) and truncated APC protein (from cancerous cells) at the cytoplasm and at the cell nucleus. Selective inhibition of PKCδ in cancer SW480 cells, which do not possess a functional ß-catenin destruction complex, did not affect ß-catenin-mediated transcriptional activity. However, in human colon carcinoma RKO cells, which have a normal ß-catenin destruction complex, negatively affected ß-catenin-mediated transcriptional activity, cell proliferation, and the expression of Wnt target genes C-MYC and CYCLIN D1. These negative effects were confirmed by siRNA-mediated knockdown of PKCδ and by the expression of a dominant negative form of PKCδ in RKO cells. Remarkably, the PKCδ stably depleted cells exhibited augmented tumorigenic activity in grafted mice. We show that PKCδ functions in a mechanism that involves regulation of ß-catenin degradation, because PKCδ inhibition induces ß-catenin stabilization at the cytoplasm and its nuclear presence at the C-MYC enhancer even without Wnt3a stimulation. In addition, expression of a dominant form of PKCδ diminished APC phosphorylation in intact cells, suggesting that PKCδ may modulate canonical Wnt activation negatively through APC phosphorylation.

Protein kinase C in Wnt signaling: implications in cancer initiation and progression.

Although it is well known that Wnt and protein kinase C (PKC) signaling pathways are both involved in carcinogenesis and tumor progression, their synergistic contribution to these processes or the crosstalk between them has just recently been approached. The Wnt and PKC signaling are involved in many cellular functions including proliferation, differentiation, survival, apoptosis, cytoskeletal remodeling, and cell motility. Canonical Wnt signaling has been well characterized as one of the most important contributors to tumorigenesis, and it has been implicated in many types of solid tumors. PKC is one of the key targets of noncanonical Wnt signaling, particularly in the Wnt/Ca(2+) pathway. Recently, data have implicated components of noncanonical Wnt/Ca(2+) and Wnt/planar cell polarity signaling in directly promoting the invasiveness and malignant progression of diverse forms of human cancer. But, unlike the canonical pathway, defining the roles of noncanonical Wnt signaling in human cancer is in its infancy. In this review, we provide a concise description of the current knowledge of the interaction between PKC and Wnt pathways and discuss the role of this crosstalk in cancer initiation and progression.

Protein kinase C ζ is a positive modulator of canonical Wnt signaling pathway in tumoral colon cell lines.

The colonic epithelium is a continuously renewing tissue with a dynamic turnover of cells. Wnt pathway is a key regulator of its homeostasis and is altered in a large proportion of colon cancers. Protein kinase C (PKC) family of serine/threonine kinases are also involved in colon tumor formation and progression; however, the molecular role played by them in the Wnt pathway, is poorly understood. Reciprocal coimmunoprecipitation and immunofluorescence studies revealed that PKCζ interacts with ß-catenin mainly in tumoral colon cells, which overexpressed this PKC isoform. The pharmacological inhibition, the small interference RNA-mediated knockdown of PKCζ or the expression of a dominant-negative form of it in tumoral SW480 cells, blocked in a dose-dependent manner the constitutive transcriptional activity mediated by ß-catenin, the cell proliferation and the expression of the Wnt target gene c-myc. Remarkably, the PKCζ stably depleted cells exhibited diminished tumorigenic activity in grafted mice. We show that PKCζ functions in a mechanism that does not involve ß-catenin degradation since the effects produced by PKCζ inhibition were also obtained in the presence of proteosome inhibitor and in cells expressing a ß-catenin degradation-resistant mutant. It was found that PKCζ activity regulates the nuclear localization of ß-catenin since PKCζ inhibition induces a rapid export of ß-catenin from the nucleus to the cytoplasm in a Leptomycin B sensitive manner. Taken together, our results indicate that the atypical PKCζ plays an important role in the positive regulation of canonical Wnt pathway.