Raúl Rincón

PP2A Inhibition Is a Common Event in Colorectal Cancer and Its Restoration Using FTY720 Shows Promising Therapeutic Potential

Protein phosphatase 2A (PP2A) is a tumor suppressor that regulates many signaling pathways crucial for cell transformation. In fact, decreased activity of PP2A has been reported as a recurrent alteration in many types of cancer. Here, we show that PP2A is frequently inactivated in patients with colorectal cancer, indicating that PP2A represents a potential therapeutic target for this disease. We identified overexpression of the endogenous PP2A inhibitors SET and CIP2A, and downregulation of regulatory PP2A such as PPP2R2A and PPP2R5E, as contributing mechanisms to PP2A inhibition in colorectal cancer. Moreover, we observed that its restoration using FTY720 impairs proliferation and clonogenic potential of colorectal cancer cells, induces caspase-dependent apoptosis, and affects AKT and extracellular signal-regulated kinase-1/2 activation status. Interestingly, treatment with FTY720 showed an additive effect with 5-fluorouracil, SN-38, and oxaliplatin, drugs used in standard chemotherapy in patients with colorectal cancer. These results suggest that PP2A activity is commonly decreased in colorectal cancer cells, and that the use of PP2A activators, such as FTY720, might represent a potential novel therapeutic strategy in colorectal cancer. Mol Cancer Ther; 13(4); 938–47. ©2014 AACR.

Deregulation of the PP2A Inhibitor SET Shows Promising Therapeutic Implications and Determines Poor Clinical Outcome in Patients with Metastatic Colorectal Cancer

Purpose: SET is an endogenous PP2A inhibitor that might represent a novel molecular target for antitumor therapy. The aim of this study was to evaluate the molecular effects of SET deregulation and its potential clinical significance in metastatic colorectal cancer (mCRC). Experimental Design: We studied the biologic effects of SET on cell growth, colonosphere formation, caspase activity, PP2A activation status, and sensitivity to oxaliplatin and FTY720 treatments. Moreover, we analyzed SET expression by immunostaining in 242 patients with mCRC. Results: SET deregulation promotes cell growth and colonosphere formation and inhibits PP2A, thereby impairing its antitumor effects. Moreover, SET reduces sensitivity to oxaliplatin in colorectal cancer cell lines, which is restored after FTY720 treatment. SET overexpression was detected in 24.8% (60 of 242) of patients with mCRC and determined significantly shorter overall (8.6 vs. 27 months; P < 0.001) and progression-free survival (7.1 vs. 13.7 months; P < 0.001), and poor response to oxaliplatin-based chemotherapy (P = 0.004). Interestingly, its prognostic value was particularly evident in patients younger than 70 years and in those harboring KRAS mutations. Conclusions: SET overexpression is a frequent event in mCRC that plays a potential oncogenic role associated with worse outcome and resistance to oxaliplatin. Moreover, this alteration defines a subgroup of patients who could benefit from therapies containing PP2A activators such as FTY720. Clin Cancer Res; 21(2); 347–56. ©2014 AACR.

Hyperphosphorylation of PP2A in colorectal cancer and the potential therapeutic value showed by its forskolin-induced dephosphorylation and activation

BACKGROUND The tumor suppressor protein phosphatase 2A (PP2A) is frequently inactivated in human cancer and phosphorylation of its catalytic subunit (p-PP2A-C) at tyrosine-307 (Y307) has been described to inhibit this phosphatase. However, its molecular and clinical relevance in colorectal cancer (CRC) remains unclear. METHODS p-PP2A-C Y307 was determined by immunoblotting in 7 CRC cell lines and 35 CRC patients. CRC cells were treated with the PP2A activator forskolin alone or combined with the PP2A inhibitor okadaic acid, 5-fluorouracil and oxaliplatin. We examined cell growth, colonosphere formation, caspase activity and AKT and ERK activation. RESULTS PP2A-C was found hyperphosphorylated in CRC cell lines. Forskolin dephosphorylated and activated PP2A, impairing proliferation and colonosphere formation, and inducing activation of caspase 3/7 and changes in AKT and ERK phosphorylation. Moreover, forskolin showed additive effects with 5-fluorouracil and oxaliplatin treatments. Analysis of p-PP2A-C Y307 in primary tumors confirmed the presence of this alteration in a subgroup of CRC patients. CONCLUSIONS Our data show that PP2A-C hyperphosphorylation is a frequent event that contributes to PP2A inhibition in CRC. Antitumoral effects of forskolin-mediated PP2A activation suggest that the analysis of p-PP2A-C Y307 status could be used to identify a subgroup of patients who would benefit from treatments based on PP2A activators.

Deregulation of miR-200b, miR-200c and miR-429 Indicates its Potential Relevant Role in Patients with Colorectal Cancer Liver Metastasis

ION CRISTÓBAL, PhD,* RAÚL RINCÓN, BSc, REBECA MANSO, BSc, CRISTINA CARAMÉS, OSCAR AGUILERA, PhD,* JUAN MADOZ-GURPIDE, PhD, FEDERICO ROJO, MD, PhD, AND JESÚS GARCÍA-FONCILLAS, MD, PhD* Translational Oncology Division, Oncohealth Institute, IIS-Fundacion Jimenez Diaz-UAM, University Hospital “Fundacion Jimenez Diaz”, Madrid, Spain Pathology Department, IIS-Fundacion Jimenez Diaz-UAM, University Hospital “Fundacion Jimenez Diaz”, Madrid, Spain

Downregulation of microRNA-199b predicts unfavorable prognosis and emerges as a novel therapeutic target which contributes to PP2A inhibition in metastatic colorectal cancer

The tumor suppressor microRNA-199b (miR-199b) is a negative SET regulator associated with poor outcome in some human cancers. However, its expression levels as well as potential biological and clinical significance in colorectal cancer (CRC) remain completely unexplored. The PP2A inhibitor SET has shown promising therapeutic and clinical implications in metastatic CRC (mCRC) but the molecular mechanisms underlying SET deregulation are currently unknown. We show here miR-199b downregulation in 4 out of 5 CRC SET-overexpressing cell lines and its inverse correlation with SET overexpression in CRC patients. Moreover, miR-199b led to PP2A activation through a direct SET inhibition, impaired cell viability and enhanced oxaliplatin sensitivity in CRC cells. MiR-199b was found downregulated in 25% of cases, and associated with lymph metastasis (p = 0.049), presence of synchronous metastasis at diagnosis (p = 0.026) and SET overexpression (p < 0.001). Furthermore, low miR-199b levels determined shorter overall (p < 0.001), progression-free survival (p = 0.003) and predicted clinical benefit to oxaliplatin treatment. The miR-199b prognostic impact was particularly evident in both younger and KRAS wild-type subgroups. Multivariate analyses confirmed its independent prognostic impact. Altogether, our results show that miR-199b is a tumor suppressor whose downregulation independently determines worse outcome and emerges as a potential contributing mechanism to inhibit PP2A via SET overexpression in a subgroup of mCRC patients.

c-Jun N-Terminal Kinase Inactivation by Mitogen-Activated Protein Kinase Phosphatase 1 Determines Resistance to Taxanes and Anthracyclines in Breast Cancer

MAPK phosphatase-1 (MKP-1) is overexpressed during malignant transformation of the breast in many patients, and it is usually associated with chemoresistance through interference with JNK-driven apoptotic pathways. Although the molecular settings of the mechanism have been documented, details about the contribution of MKP-1 to the failure of chemotherapeutic interventions are unclear. Transient overexpression of MKP-1 and treatment with JNK-modulating agents in breast carcinoma cells confirmed the mediation of MKP-1 in the resistance to taxanes and anthracyclines in breast cancer, through the inactivation of JNK1/2. We next assessed MKP-1 expression and JNK1/2 phosphorylation status in a large cohort of samples from 350 early breast cancer patients treated with adjuvant anthracycline–based chemotherapy. We detected that MKP-1 overexpression is a recurrent event predominantly linked to dephosphorylation of JNK1/2 with an adverse impact on relapse of the tumor and overall and disease-free survival. Moreover, MKP-1 and p-JNK1/2 determinations in 64 locally advanced breast cancer patients treated with neoadjuvant taxane–based chemotherapy showed an inverse correlation between MKP-1 overexpression (together with JNK1/2 inhibition) and the pathologic response of the tumors. Our results emphasize the importance of MKP-1 as a potential predictive biomarker for a subset of breast cancer patients with worse outcome and less susceptibility to treatment. Mol Cancer Ther; 15(11); 2780–90. ©2016 AACR.

Up-regulation of c-Cbl suggests its potential role as oncogene in primary colorectal cancer

Dear Editor: Although progressive advances in early detection has been carried out in the last years, colorectal cancer (CRC) still represents the third cause of cancer death worldwide. This is because more than half of the patients progress to metastatic disease, which determines a very poor outcome. Therefore, it remains necessary to better understand the molecular basis that governs the progression from the early stages of CRC to metastatic development. In this context, the acquisition of invasiveness properties is a key molecular event that determines the progression from primary tumor to metastasis. Of importance, it has been recently reported that c-Cbl, an E3 ubuquitin ligase and a multifunctional adaptor protein, regulates invasion ability of cancer cells through matrix metalloproteinase 2. It is an unexpected observation since c-Cbl was largely reported to play a tumor suppressor role in several tumor types. However, in concordance with this novelproposed oncogenic role as regulator of cell adhesion, migration, and degradation, c-Cbl has been described as a marker of poor clinical outcome in prostate cancer. Therefore, it seems that this protein could be playing a dual role as a tumor suppressor or oncogene depending on the cancer model and stage of the disease. This novel-reported function for c-Cbl and the fact that its role in CRC remains unclear prompted us to analyze c-Cbl expression by western blot in a series of seven CRC cell lines. Interestingly, we observed increased c-Cbl levels in the DLD-1, RKO, LoVo, and SW620 cell lines whereas the WiDr, SW480, and HT-29 cell lines showed a c-Cbl expression similar to normal colonic mucosa. To further evaluate the importance of in CRC, we studied c-Cbl expression levels in 22 patients with primary CRC. Primary colorectal tissues were surgical resection specimens from CRC tumors obtained from Fundacion Jimenez Diaz Biobank (BFJD, Madrid). Tumor, node, metastases (TNM) staging was classified using the 7th American Joint Committee on Cancer (AJCC) staging system for colorectal cancer. Clinical data were collected from medical clinical records by an oncologist (J. G.-F.). Paired normal mucosa obtained from each patient was used as control. Moreover, a pathologist confirmed that primary tumor tissues used in this work contained greater than 70 % tumoral component (F. R.). All samples were taken anonymously and the ethical committee and institutional review board approved the project. Importantly, we found that 8 out of the 22 CRC cases showed c-Cbl overexpression in the tumor samples compared to their paired normal colonic mucosa. These results confirmed the previous observations made in CRC cell lines. Altogether, our findings suggest that c-Cbl deregulation is a recurrent event that could be playing a role in the acquisition of invasiveness properties of CRC cells and might serve as a novel potential therapeutic target in a subset of CRC patients. However, further studies are needed to clarify this potential role of c-Cbl as oncogene in primary colorectal cancer and to determine its biological and clinical significance in the pathogenesis of this disease.

Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma

Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

Potential involvement of protein phosphatase 2A in temsirolimus-mediated resensitization to cetuximab in colon cancer cells

Our group has just described that inhibition of the tumor suppressor PP2A is a frequent event in CRC and that its restoration using FtY720 induces potent antitumor effects (Cristobal et al. 2014). Furthermore, another recent work showed that FtY720 restores sensitivity to cetuximab in CRC cells with acquired resistance to this drug both in vitro and in vivo (Rosa et al. 2013). thus, taking into account these observations and the fact that we identified CIP2A as a molecular target of FtY720 (Cristobal et al. 2014), responsible as least in part of the FtY720-mediated PP2A activation that determines its antitumor activity, we wonder about the possibility that a similar signaling cascade could be involved in the effects induced by the treatment with temsirolimus. In fact, CIP2A is an endogenous PP2A inhibitor that impairs PP2A-induced c-MYC dephosphorylation, which marks this protein for ubiquitination and proteome degradation (Junttila and Westermarck 2008). It would be therefore very interesting to assess c-MYC levels after temsirolimus treatment since it represents a highly relevant oncogene in human cancer that could be playing an important role in temsirolimus anticancer activities. Moreover, in concordance with this hypothesis Wang et al. (2014) observed that temsirolimus also affects AKt and eRK phosphorylation status, both direct targets of the PP2A phosphatase activity. therefore, it would be very interesting to analyze the effects of PP2A overexpression and knockdown or the use of a PP2A inhibitor such as okadaic acid to further confirm that temsirolimus-induced sensitivity of CRC cells is dependent at least partially on PP2A activation. In conclusion, future studies are needed to fully clarify the role of PP2A in the molecular mechanism by which temsirolimus is acting, and its potential relevance in an acquisition of resistance to cetuximab in CRC cells. We read with great interest the work recently published by Wang et al. (2014) which provides experimental evidences about the synergistic effects of temsirolimus with cetuximab via down-regulation of cancerous inhibitor of protein phosphatase 2A (PP2A) (CIP2A) in colorectal cancer (CRC) cells. CIP2A is, as indicated by its own name, a potent endogenous PP2A inhibitor frequently deregulated in human cancer (Junttila et al. 2007). the authors found that temsirolimus, an inhibitor of the mammalian target of rapamycin (hudes et al. 2007), downregulates CIP2A by decreasing its transcriptional levels and enhancing protein degradation through the lysosomal autophagy pathway. Moreover, they observed that temsirolimus was able to sensitize the K-ras codon 13 mutated hCt-15 cell line to cetuximab in a mouse model. these are important findings since the development of resistance to cetuximab in patients with metastatic CRC is a very recurrent event after few months of treatment that severely compromises their clinical outcome. therefore, it would be very interesting to develop alternative therapeutic strategies that could resensitize CRC cells to cetuximab, then enhancing the period of time in which this drug is effective.