Inhibition of Multifunctional Protein p32/C1QBP Promotes Cytostatic Effects in Colon Cancer Cells by Altering Mitogenic Signaling Pathways and Promoting Mitochondrial Damage.

The protein p32 (C1QBP) is a multifunctional and multicompartmental homotrimer that is overexpressed in many cancer types, including colon cancer. High expression levels of C1QBP are negatively correlated with the survival of patients. Previously, we demonstrated that C1QBP is an essential promoter of migration, chemoresistance, clonogenic, and tumorigenic capacity in colon cancer cells. However, the mechanisms underlying these functions and the effects of specific C1QBP protein inhibitors remain unexplored. Here, we show that the specific pharmacological inhibition of C1QBP with the small molecule M36 significantly decreased the viability rate, clonogenic capacity, and proliferation rate of different colon cancer cell lines in a dose-dependent manner. The effects of the inhibitor of C1QBP were cytostatic and non-cytotoxic, inducing a decreased activation rate of critical pro-malignant and mitogenic cellular pathways such as Akt-mTOR and MAPK in RKO colon cancer cells. Additionally, treatment with M36 significantly affected the mitochondrial integrity and dynamics of malignant cells, indicating that p32/C1QBP plays an essential role in maintaining mitochondrial homeostasis. Altogether, our results reinforce that C1QBP is an important oncogene target and that M36 may be a promising therapeutic drug for the treatment of colon cancer.

Canonical Wnt Pathway Is Involved in Chemoresistance and Cell Cycle Arrest Induction in Colon Cancer Cell Line Spheroids.

The presence of cancer stem cells (CSCs) has been associated with the induction of drug resistance and disease recurrence after therapy. 5-Fluorouracil (5FU) is widely used as the first-line treatment of colorectal cancer (CRC). However, its effectiveness may be limited by the induction of drug resistance in tumor cells. The Wnt pathway plays a key role in the development and CRC progression, but it is not clearly established how it is involved in CSCs resistance to treatment. This work aimed to investigate the role played by the canonical Wnt/ß-catenin pathway in CSCs resistance to 5FU treatment. Using tumor spheroids as a model of CSCs enrichment of CRC cell lines with different Wnt/ß-catenin contexts, we found that 5FU induces in all CRC spheroids tested cell death, DNA damage, and quiescence, but in different proportions for each one: RKO spheroids were very sensitive to 5FU, while SW480 were less susceptible, and the SW620 spheroids, the metastatic derivative of SW480 cells, displayed the highest resistance to death, high clonogenic capacity, and the highest ability for regrowth after 5FU treatment. Activating the canonical Wnt pathway with Wnt3a in RKO spheroids decreased the 5FU-induced cell death. But the Wnt/ß-catenin pathway inhibition with Adavivint alone or in combination with 5FU in spheroids with aberrant activation of this pathway produced a severe cytostatic effect compromising their clonogenic capacity and diminishing the stem cell markers expression. Remarkably, this combined treatment also induced the survival of a small cell subpopulation that could exit the arrest, recover SOX2 levels, and re-grow after treatment.

Non-canonical Wnt/Ca2+ signaling is essential to promote self-renewal and proliferation in colon cancer stem cells.

Introduction: Cancer Stem Cells (CSC) are responsible for maintaining tumor growth, chemoresistance, and metastasis. Therefore, understanding their characteristics is critical to progress in cancer therapy. While the contribution of the canonical Wnt/b-catenin signaling in both normal and CSCs had been well established, the function of non-canonical Wnt signaling cascades in stem cells is unclear. Recently, we reported that Wnt ligands trigger complex signaling in which the canonical and non-canonical responses can be simultaneously activated by one ligand in colon cancer cells, suggesting, therefore, that noncanonical Wnt pathways may also be important in CSCs. Methods: The present work aimed to know the role of the Wnt/Ca2+ pathway in colon CSCs. We used tumorspheres as a model of CSCs enrichment of CRC cell lines with different Wnt/b-catenin contexts. Results: Using Wnt3a and Wnt5a as prototype ligands to activate the canonical or the non-canonical pathways, respectively, we found that both Wnt3a and Wnt5a promote sphere-formation capacity and proliferation without stimulating b-catenin-dependent transcription. Upregulation of sphere formation by Wnt5a or Wnt3a requires the downstream activation of Phospholipase C and transcriptional factor NFAT. Moreover, the single specific inhibition of PLC or NFAT, using U73122 and 11R-VIVIT, respectively, leads to impaired sphere formation. Discussion: Our results indicate that both types of ligands activate the Wnt/Ca2+ signaling axis to induce/maintain the self-renewal efficiency of CSCs, demonstrating to be essential for the functions of CSC in colon cancer.

Hypoxia-inducible factors, mTOR, and astrin constitute an integrative regulatory network in colon cancer cells.

Astrin/SPAG5 is a mitotic spindle protein found to be overexpressed in several human cancers, functioning as an oncogene. The expression of Astrin has not been reported so far in colon cancer, nor has it been related to HIFs expression or action. Since mTOR, Astrin, and hypoxia-inducible factors (HIFs) are involved in promoting the growth and survival of cancer cells, we investigated the possible interaction between them in cultured colon cancer cells. Both Astrin and HIF-1α and HIF-2α protein levels were found only expressed in colon cancer cells compared with nonmalignant cells. Our data indicate that mTOR stimulates both Astrin and HIFs expression, but notably, mTORC activity seems to be independent of Astrin expression levels. However, while HIF-1α or HIF-2α stable knockdown increased Astrin expression, mTOR activity was affected in an opposite way by HIF-1α or HIF-2α silencing, indicating that HIF-1α inhibits mTOR while HIF-2α stimulates its activity. These data suggest that mTOR, Astrin, and HIFs compose an integrative network interacting to activate positive or negative regulatory loops probably to coordinate cancer cell growth, metabolism, and survival under oncogenic stress.

Increased O-GlcNAcylation promotes IGF-1 receptor/PhosphatidyI Inositol-3 kinase/Akt pathway in cervical cancer cells.

O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification on serine and threonine residues of cytosolic, nuclear and mitochondrial proteins. O-GlcNAcylation level is regulated by OGT (O-GlcNAc transferase), which adds GlcNAc on proteins, and OGA (O-GlcNAcase), which removes it. Abnormal level of protein O-GlcNAcylation has been observed in numerous cancer cell types, including cervical cancer cells. In the present study, we have evaluated the effect of increasing protein O-GlcNAcylation on cervical cancer-derived CaSki cells. We observed that pharmacological enhancement of protein O-GlcNAcylation by Thiamet G (an inhibitor of OGA) and glucosamine (which provides UDP-GlcNAc substrate to OGT) increases CaSki cells proliferation, migration and survival. Moreover, we showed that increased O-GlcNAcylation promotes IGF-1 receptor (IGF1R) autophosphorylation, possibly through inhibition of protein tyrosine-phosphatase 1B activity. This was associated with increased IGF-1-induced phosphatidyl-Inositol 3-phosphate production at the plasma membrane and increased Akt activation in CaSki cells. Finally, we showed that protein O-GlcNAcylation and Akt phosphorylation levels were higher in human cervical cancer samples compared to healthy cervix tissues, and a highly positive correlation was observed between O-GlcNAcylation level and Akt phosphorylation in theses tissues. Together, our results indicate that increased O-GlcNAcylation, by activating IGF1R/ Phosphatidyl inositol 3-Kinase (PI-3K)/Akt signaling, may participate in cervical cancer cell growth and proliferation.