Mexican Colorectal Cancer Research Consortium (MEX-CCRC): Etiology, Diagnosis/Prognosis, and Innovative Therapies.

In 2013, recognizing that Colorectal Cancer (CRC) is the second leading cause of death by cancer worldwide and that it was a neglected disease increasing rapidly in Mexico, the community of researchers at the Biomedicine Research Unit of the Facultad de Estudios Superiores Iztacala from the Universidad Nacional Autónoma de México (UNAM) established an intramural consortium that involves a multidisciplinary group of researchers, technicians, and postgraduate students to contribute to the understanding of this pathology in Mexico. This article is about the work developed by the Mexican Colorectal Cancer Research Consortium (MEX-CCRC): how the Consortium was created, its members, and its short- and long-term goals. Moreover, it is a narrative of the accomplishments of this project. Finally, we reflect on possible strategies against CRC in Mexico and contrast all the data presented with another international strategy to prevent and treat CRC. We believe that the Consortium's characteristics must be maintained to initiate a national strategy, and the reported data could be useful to establish future collaborations with other countries in Latin America and the world.

Recent advances in PTP1B signaling in metabolism and cancer.

Protein tyrosine phosphorylation is one of the major post-translational modifications in eukaryotic cells and represents a critical regulatory mechanism of a wide variety of signaling pathways. Aberrant protein tyrosine phosphorylation has been linked to various diseases, including metabolic disorders and cancer. Few years ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors, able to block the signals emanating from receptor tyrosine kinases. However, recent evidence demonstrates that misregulation of PTPs activity plays a critical role in cancer development and progression. Here, we will focus on PTP1B, an enzyme that has been linked to the development of type 2 diabetes and obesity through the regulation of insulin and leptin signaling, and with a promoting role in the development of different types of cancer through the activation of several pro-survival signaling pathways. In this review, we discuss the molecular aspects that support the crucial role of PTP1B in different cellular processes underlying diabetes, obesity and cancer progression, and its visualization as a promising therapeutic target.

p21-Activated Kinase 1 Promotes Breast Tumorigenesis via Phosphorylation and Activation of the Calcium/Calmodulin-Dependent Protein Kinase II.

p21-Activated kinase-1 (Pak1) is frequently overexpressed and/or amplified in human breast cancer and is necessary for transformation of mammary epithelial cells. Here, we show that Pak1 interacts with and phosphorylates the Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), and that pharmacological inhibition or depletion of Pak1 leads to diminished activity of CaMKII. We found a strong correlation between Pak1 and CaMKII expression in human breast cancer samples, and combined inhibition of Pak1 and CaMKII with small-molecule inhibitors was synergistic and induced apoptosis more potently in Her2 positive and triple negative breast cancer (TNBC) cells. Co-adminstration of Pak and CaMKII small-molecule inhibitors resulted in a dramatic reduction of proliferation and an increase in apoptosis in a 3D cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic strategies in breast cancer.

IFI27/ISG12 Downregulates Estrogen Receptor α Transactivation by Facilitating Its Interaction With CRM1/XPO1 in Breast Cancer Cells.

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor whose activity is modulated by its interaction with multiple protein complexes. In this work, we have identified the protein interferon alpha inducible protein 27 (IFI27/ISG12) as a novel ERα-associated protein. IFI27/ISG12 transcription is regulated by interferon and estradiol and its overexpression is associated to reduced overall survival in ER+ breast cancer patients but its function in mammary gland tissue remains elusive. In this study we showed that overexpression of IFI27/ISG12 in breast cancer cells attenuates ERα transactivation activity and the expression of ERα-dependent genes. Our results demonstrated that IFI27/ISG12 overexpression in MCF-7 cells reduced their proliferation rate in 2-D and 3-D cell culture assays and impaired their ability to migrate in a wound-healing assay. We show that IFI27/ISG12 downregulation of ERα transactivation activity is mediated by its ability to facilitate the interaction between ERα and CRM1/XPO1 that mediates the nuclear export of large macromolecules to the cytoplasm. IFI27/ISG12 overexpression was shown to impair the estradiol-dependent proliferation and tamoxifen-induced apoptosis in breast cancer cells. Our results suggest that IFI27/ISG12 may be an important factor in regulating ERα activity in breast cancer cells by modifying its nuclear versus cytoplasmic protein levels. We propose that IFI27/ISG12 may be a potential target of future strategies to control the growth and proliferation of ERα-positive breast cancer tumors.

Helminth-derived molecules inhibit colitis-associated colon cancer development through NF-κB and STAT3 regulation.

Inflammation is currently considered a hallmark of cancer and plays a decisive role in different stages of tumorigenesis, including initiation, promotion, progression, metastasis and resistance to antitumor therapies. Colorectal cancer is a disease widely associated with local chronic inflammation. Additionally, extrinsic factors such as infection may beneficially or detrimentally alter cancer progression. Several reports have noted the ability of various parasitic infections to modulate cancer development, favoring tumor progression in many cases and inhibiting tumorigenesis in others. The aim of our study was to determine the effects of excreted/secreted products of the helminth Taenia crassiceps (TcES) as a treatment in a murine model of colitis-associated colon cancer (CAC). Here, we found that after inducing CAC, treatment with TcES was able to reduce inflammatory cytokines such as IL-1ß, TNF-α, IL-33 and IL-17 and significantly attenuate colon tumorigenesis. This effect was associated with the inhibition of signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation. Furthermore, we determined that TcES interfered with LPS-induced NF-κB p65 activation in human colonic epithelial cell lines in a Raf-1 proto-oncogene-dependent manner. Moreover, in three-dimensional cultures, TcES promoted reorganization of the actin cytoskeleton, altering cell morphology and forming colonospheres, features associated with a low grade of aggressiveness. Our study demonstrates a remarkable effect of helminth-derived molecules on suppressing ongoing colorectal cancer by downregulating proinflammatory and protumorigenic signaling pathways.

Early and Partial Reduction in CD4+Foxp3+ Regulatory T Cells during Colitis-Associated Colon Cancer Induces CD4+ and CD8+ T Cell Activation Inhibiting Tumorigenesis.

Colorectal cancer (CRC) is the second most commonly diagnosed cancer in women and the third in men in North America and Europe. CRC is associated with inflammatory responses in which intestinal pathology is caused by different cell populations including a T cell dysregulation that concludes in an imbalance between activated T (Tact) and regulatory T (Treg) cells. Treg cells are CD4+Foxp3+ cells that actively suppress pathological and physiological immune responses, contributing to the maintenance of immune homeostasis. A tumor-promoting function for Treg cells has been suggested in CRC, but the kinetics of Treg cells during CRC development are poorly known. Therefore, using a mouse model of colitis-associated colon cancer (CAC) induced by azoxymethane and dextran sodium sulfate, we observed the dynamic and differential kinetics of Treg cells in blood, spleen and mesenteric lymph nodes (MLNs) as CAC progresses, highlighting a significant reduction in Treg cells in blood and spleen during early CAC development, whereas increasing percentages of Treg cells were detected in late stages in MLNs. Interestingly, when Treg cells were decreased, Tact cells were increased and vice versa. Treg cells from late stages of CAC displayed an activated phenotype by expressing PD1, CD127 and Tim-3, suggesting an increased suppressive capacity. Suppression assays showed that T-CD4+ and T-CD8+ cells were suppressed more efficiently by MLN Treg cells from CAC animals. Finally, an antibody-mediated reduction in Treg cells during early CAC development resulted in a better prognostic value, because animals showed a reduction in tumor progression associated with an increased percentage of activated CD4+CD25+Foxp3- and CD8+CD25+ T cells in MLNs, suggesting that Treg cells suppress T cell activation at early steps during CAC development.

Lack of STAT6 Attenuates Inflammation and Drives Protection against Early Steps of Colitis-Associated Colon Cancer.

Colitis-associated colon cancer (CAC) is one of the most common malignant neoplasms and a leading cause of death. The immunologic factors associated with CAC development are not completely understood. Signal transducer and activator of transcription 6 (STAT6) is part of an important signaling pathway for modulating intestinal immune function and homeostasis. However, the role of STAT6 in colon cancer progression is unclear. Following CAC induction in wild-type (WT) and STAT6-deficient mice (STAT6-/-), we found that 70% of STAT6-/- mice were tumor-free after 8 weeks, whereas 100% of WT mice developed tumors. STAT6-/- mice displayed fewer and smaller colorectal tumors than WT mice; this reduced tumorigenicity was associated with decreased proliferation and increased apoptosis in the colonic mucosa in the early steps of tumor progression. STAT6-/- mice also exhibited reduced inflammation, diminished concentrations COX2 and nuclear ß-catenin protein in the colon, and decreased mRNA expression of IL17A and TNFα, but increased IL10 expression when compared with WT mice. Impaired mucosal expression of CCL9, CCL25, and CXCR2 was also observed. In addition, the number of circulating CD11b+Ly6ChiCCR2+ monocytes and CD11b+Ly6ClowLy6G+ granulocytes was both decreased in a STAT6-dependent manner. Finally, WT mice receiving a STAT6 inhibitor in vivo confirmed a significant reduction in tumor load as well as less intense signs of CAC. Our results demonstrate that STAT6 is critical in the early steps of CAC development for modulating inflammatory responses and controlling cell recruitment and proliferation. Thus, STAT6 may represent a promising target for CAC treatment. Cancer Immunol Res; 5(5); 385-96. ©2017 AACR.

Reduced PAK1 activity sensitizes FA/BRCA-proficient breast cancer cells to PARP inhibition.

Cells that are deficient in homologous recombination, such as those that have mutations in any of the Fanconi Anemia (FA)/BRCA genes, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, FA/BRCA-deficient tumors represent a small fraction of breast cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. The gene encoding the serine-threonine protein kinase p21-activated kinase 1 (PAK1) is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme controls many cellular processes by phosphorylating both cytoplasmic and nuclear substrates. Here, we show that depletion or pharmacological inhibition of PAK1 down-regulated the expression of genes involved in the FA/BRCA pathway and compromised the ability of cells to repair DNA by Homologous Recombination (HR), promoting apoptosis and reducing colony formation. Combined inhibition of PAK1 and PARP in PAK1 overexpressing breast cancer cells had a synergistic effect, enhancing apoptosis, suppressing colony formation, and delaying tumor growth in a xenograft setting. Because reduced PAK1 activity impaired FA/BRCA function, inhibition of this kinase in PAK1 amplified and/or overexpressing breast cancer cells represents a plausible strategy for expanding the utility of PARP inhibitors to FA/BRCA-proficient cancers.

Anti-inflammatory and Antitumor Activity of a Triple Therapy for a Colitis-Related Colorectal Cancer.

Colorectal cancer (CRC) is an important health issue worldwide, accounting for the third place of cancer incidence. Chronic inflammation, as seen in Crohn's disease and ulcerative colitis, is the most important risk factor for developing CRC, as it favours neoplastic transformation by enhancing epithelial cell turnover in the colonic mucosa. Treatments for CRC need to be improved; currently they are not specific and have several secondary effects in patients. The main objective of this work was to evaluate a new therapeutic strategy against a colitis-related colorectal cancer in vivo and in vitro by targeting mTOR-signaling and lactate dehydrogenase A. Together, these mechanisms directly affect tumor energetics. In this study we evaluated a better and more efficient triple therapy against a chronic inflammation-associated CRC in vivo and in vitro. After the development of tumors, mice were treated intraperitoneally during a forty-day period with single drugs or different combinations of Metformin, Sodium Oxamate and Doxorubicin. Targeted inhibition of the mTOR pathway, lactate dehydrogenase A and the concurrent use of Doxorubicin (called in this work as triple therapy), leaded to a notable reduction in the number and size of tumors in mice, and, a significant pro-inflammatory cytokines reduction Besides, we showed that treated cells were induced to early autophagy, and apoptosis cell death. Our results represent a novel and robust therapeutic strategy for overcoming CRC by means of targeting central molecular pathways in cancer by the combination of Metformin, Oxamate, and Doxorubicin leading to a rapid tumor growth inhibition and a dramatic colorectal crypt restoration. Besides, drug combination resulted in a notable reduction of anti-inflammatory cytokines.

H-ras Inhibits the Hippo Pathway by Promoting Mst1/Mst2 Heterodimerization.

The protein kinases Mst1 and Mst2 have tumor suppressor activity, but their mode of regulation is not well established. Mst1 and Mst2 are broadly expressed and may have certain overlapping functions in mammals, as deletions of both Mst1 and Mst2 together are required for tumorigenesis in mouse models [1-3]. These kinases act via a three-component signaling cascade comprising Mst1 and Mst2, the protein kinases Lats1 and Lats2, and the transcriptional coactivators Yap and Taz [4-6]. Mst1 and Mst2 contain C-terminal SARAH domains that mediate their homodimerization as well as heterodimerization with other SARAH domain-containing proteins, which may regulate Mst1/Mst2 activity. Here we show that, in addition to forming homodimers, Mst1 and Mst2 heterodimerize in cells, this interaction is mediated by their SARAH domains and is favored over homodimers, and these heterodimers have much-reduced protein kinase activity compared to Mst1 or Mst2 homodimers. Mst1/Mst2 heterodimerization is strongly promoted by oncogenic H-ras, and this effect requires activation of the Erk pathway. Cells lacking Mst1, in which Mst1/Mst2 heterodimers are not possible, are resistant to H-ras-mediated transformation and maintain active hippo pathway signaling compared to wild-type cells or cells lacking both Mst1 and Mst2. Our results suggest that H-ras, via an Erk-dependent mechanism, downregulates Mst1/Mst2 activity by inducing the formation of inactive Mst1/Mst2 heterodimers.

Targeting Cdc42 in cancer.

INTRODUCTION: The Rho GTPases are a family of proteins that control fundamental cellular processes in response to extracellular stimuli and internal programs. Rho GTPases function as molecular switches in which the GTP-bound proteins are active and GDP-bound proteins are inactive. This article will focus on one Rho family member, Cdc42, which is overexpressed in a number of human cancers, and which might provide new therapeutic targets in malignancies. AREAS COVERED: In this article, the key regulators and effectors of Cdc42 and their molecular alterations are described. The complex interactions between the signaling cascades regulated by Cdc42 are also analyzed. EXPERT OPINION: While mutations in Cdc42 have not been reported in human cancer, aberrant expression of Cdc42 has been reported in a variety of tumor types and in some instances has been correlated with poor prognosis. Recently, it has been shown that Cdc42 activation by oncogenic Ras is crucial for Ras-mediated tumorigenesis, suggesting that targeting Cdc42 or its effectors might be useful in tumors harboring activating Ras mutations.

Pak1 kinase links ErbB2 to ß-catenin in transformation of breast epithelial cells.

p21-Activated kinase-1 (Pak1) is frequently upregulated in human breast cancer and is required for transformation of mammary epithelial cells by ErbB2. Here, we show that loss of Pak1, but not the closely related Pak2, leads to diminished expression of ß-catenin and its target genes. In MMTV-ErbB2 transgenic mice, loss of Pak1 prolonged survival, and mammary tissues of such mice showed loss of ß-catenin. Expression of a ß-catenin mutant bearing a phospho-mimetic mutation at Ser 675, a specific Pak1 phosphorylation site, restored transformation to ErbB2-positive, Pak1-deficient mammary epithelial cells. Mice bearing xenografts of ErbB2-positive breast cancer cells showed tumor regression when treated with small-molecule inhibitors of Pak or ß-catenin, and combined inhibition by both agents was synergistic. These data delineate a signaling pathway from ErbB2 to Pak to ß-catenin that is required for efficient transformation of mammary epithelial cells, and suggest new therapeutic strategies in ErbB2-positive breast cancer.

p21-activated kinases in Erbb2-positive breast cancer: A new therapeutic target?

The activation of receptor tyrosine kinases, particularly ErbB2, has been linked to the genesis and progression of breast cancer. Two of the central signaling pathways activated by ErbB2 are the Ras/Raf-1/Mek/Erk pathway, which plays an important role in tumor cell growth and migration, and the PI3K/Akt pathway, which plays an important role in cell survival. Recently, we and others have shown that signaling through the Ras-Erk pathway can be influenced by p21-activated kinase 1 (Pak1), an effector of the Rho family GTP ases Rac and Cdc42. Expression of activated forms of Rac promotes activation of Erk through mechanisms involving Pak1 phosphorylation of Raf-1 and Mek1. In addition, Pak1 has also been implicated in the activation of Akt. However, our understanding regarding the degree to which Rho GTPases, and their effectors such as Pak1, contribute to ErbB2-mediated signaling is very limited.Recent results from our laboratory indicate that ErbB2 expression correlates with Pak activation in estrogen receptor negative human breast tumor samples. Using a three-dimensional (3D) culture of human MCF-10A mammary epithelial cells, we found that activation of Rac-Pak pathway by ErbB2 induces growth factor independent proliferation and promotes disruption of acini-like structures through the activation of the Erk and Akt pathways. We also observed that blocking Pak1 activity by small molecule inhibitors impeded the ability of activated ErbB2 to transform these cells and to activate its associated downstream signaling targets. In addition, we found that suppressing Pak activity in ErbB2-amplified breast cancer cells delayed tumor formation and downregulated Erk and Akt signaling in vivo. These results support a model in which Pak, by activating Erk and Akt, cooperates with ErbB2 in transforming mammary epithelial cells.

Interaction with LC8 is required for Pak1 nuclear import and is indispensable for zebrafish development.

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

Activation of Src by protein tyrosine phosphatase 1B Is required for ErbB2 transformation of human breast epithelial cells.

Protein tyrosine phosphatase (PTP) 1B plays a major role in inhibiting signaling from the insulin and leptin receptors. Recently, PTP1B was found to have an unexpected positive role in ErbB2 signaling in a mouse model of breast cancer, but the mechanism underlying this effect has been unclear. Using human breast epithelial cells grown in a three-dimensional matrix, we found that PTP1B, but not the closely related enzyme T-cell PTP, is required for ErbB2 transformation in vitro. Activation of ErbB2, but not ErbB1, increases PTP1B expression, and increased expression of PTP1B activates Src and induces a Src-dependent transformed phenotype. These findings identify a molecular mechanism by which PTP1B links an important oncogenic receptor tyrosine kinase to signaling pathways that promote aberrant cell division and survival in human breast epithelial cells.

A tale of two Paks.

Paks (p21-activated kinases) are effectors for the small GTPases Cdc42 (cell division cycle 42) and Rac that play fundamental roles in a wide range of cellular processes, including cell morphology, motility, survival, gene transcription, apoptosis and hormone signalling. These enzymes are widely expressed in numerous tissues and are activated by extracellular signals through GTPase-dependent and -independent mechanisms. On the basis of structural and biochemical features, the Pak family members have been classified into two groups, comprising three members each. The two Pak groups have generally been considered as two halves of a single entity, but accumulating evidence indicates that this is not so. In this review, we discuss recent advances in our understanding of the structure, regulation and function of these kinases, emphasizing the many differences between these two groups of signalling proteins.

EhGEF3, a novel Dbl family member, regulates EhRacA activation during chemotaxis and capping in Entamoeba histolytica.

Rho GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to cytoskeleton. The Rho guanine nucleotide exchange factors (RhoGEFs) have been implicated in direct activation of these GTPases. Here, we describe a novel RhoGEF, denominated EhGEF3 from the parasite Entamoeba histolytica, which encodes a 110 kDa protein containing the domain arrangement of a Dbl homology domain in tandem with a pleckstrin homology domain, the DH domain of EhGEF3 is closely related with the one of the Vav3 protein. Biochemical analysis revealed that EhGEF3 is capable of stimulating nucleotide exchange on the E. histolytica EhRacA and EhRho1 GTPases in vitro, however only a partial GEF activity toward Cdc42 was observed. Conserved residue analysis showed that the N816 and L817 residues are critical for EhGEF3 activity. Cellular studies revealed that EhGEF3 colocalises with EhRacA in the rear of migrating cells, probably regulating the retraction of the uroid and promoting the activation of these GTPase during the chemotactic response toward fibronectin, and that EhGEF3 also regulates EhRacA activation during the capping of cell receptors. These results suggest that EhGEF3 should have a direct role in activating EhRacA, and in bringing the activated GTPase to specific target sites such as the uroid.