Cristina Caramés

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

Phosphorylated protein phosphatase 2A determines poor outcome in patients with metastatic colorectal cancer

Background:Protein phosphatase 2A (PP2A) is a tumour suppressor frequently inactivated in human cancer and its tyrosine-307 phosphorylation has been reported as a molecular inhibitory mechanism.Methods:Expression of phosphorylated PP2A (p-PP2A) was evaluated in 250 metastatic colorectal cancer (CRC) patients. Chi-square, Kaplan–Meier and Cox analyses were used to determine correlations with clinical and molecular parameters and impact on clinical outcomes.Results:High p-PP2A levels were found in 17.2% cases and were associated with ECOG performance status (P=0.001) and presence of synchronous metastasis at diagnosis (P=0.035). This subgroup showed substantially worse overall survival (OS) (median OS, 6.0 vs 26.2 months, P<0.001) and progression-free survival (PFS) (median PFS, 3.8 vs 13.3 months, P<0.001). The prognostic impact of p-PP2A was particularly evident in patients aged <70 years (P<0.001). Multivariate analysis revealed that p-PP2A retained its prognostic impact for OS (hazard ratio 2.7; 95% confidence interval, 1.8–4.1; P<0.001) and PFS (hazard ratio 3.0; 95% confidence interval, 1.8–5.0; P<0.001).Conclusions:Phosphorylated PP2A is an alteration that determines poor outcome in metastatic CRC and represents a novel potential therapeutic target in this disease, thus enabling to define a subgroup of patients who could benefit from future treatments based on PP2A activators.

Downregulation of miR-214 is specific of liver metastasis in colorectal cancer and could play a role determining the metastatic niche

To the Editor: MicroRNAs are small, non-coding RNAs that negatively regulate the expression of their target genes. Aberrant microRNA expression levels have been largely reported in human cancer and have emerged as novel candidates for targeted therapies. It has been recently reported that miR214 is a negative regulator of colorectal cancer (CRC) liver metastasis. Interestingly, the authors found that restored miR214 expression suppressed in vitro proliferation, migration and invasion, and tumor growth and liver metastasis in vivo, and identified FGFR1 as a miR-214 target. However, the fact that it does not clarify whether miR-214 downregulation is a molecular event specific of liver metastases or characteristic of the metastatic CRC with independence of the metastatic site is a relevant question to be addressed. This prompted us to analyzed the expression pattern of 377 mature microRNAs using Taqman low-density arrays (TLDAs) panel A (Applied Biosystems) in 17 CRC patients, 12 with liver metastasis, and 5 with lungmetastasis previously reviewed by a pathologist (F.R.) to further confirm the diagnosis. All samples were taken anonymously, and the ethical committee and institutional review board approved the project. Analysis of relative gene expression data was performed using the 2T method, and U6B was used as internal control. Thus, we found miR-214 significantly downregulated in liver metastatic tissues compared with their paired primary CRC tissues (P=0.007). Moreover, we detected higher miR214 levels in lung metastasis compared to their paired primary CRC tissues, although significance was not achieved in this case (P=0.092) and no differences were observed in miR-214 expression between the liver and lung metastatic CRC tissues (P=0.282). Interestingly, miR-214 showed significantly lower expression in primary CRC tissues from patients with lung metastasis than in those with liver metastasis (P=0.024). Altogether, our results confirm the previous reported role of miR-214 in CRC with liver metastasis and additionally indicate that miR-214 downregulation is specific of liver metastatic location. Moreover, our observations suggest that miR-214 expression in the primary CRC could be playing a potential role determining the metastatic niche although further studies are needed to clarify this point.

Decreased PLK1 expression denotes therapy resistance and unfavourable disease-free survival in rectal cancer patients receiving neoadjuvant chemoradiotherapy

AIM Polo-like kinase 1 (Plk1) plays a key role in mitotic cell division and DNA damage repair. It has been observed that either up-regulated or down-regulated Plk1 could induce mitotic defects that results in aneuploidy and tumorigenesis, probably depending on the context. Few previous reports have associated Plk1 expression with prognosis and response to radiotherapy in rectal carcinomas. The aim of this study is to investigate the prognostic impact of Plk1 expression and its role in predicting response to neoadjuvant cheomoradiotherapy in rectal cancer. METHODS AND RESULTS Immunohistochemical analysis of Plk1 expression was performed in the pre-treatment tumour specimens from 75 rectal cancer patients. We analysed the assocation between Plk1 expression and clinicopathological parameters, pathologic response and outcome. Opposed to previous reports on this issue, low expression of Plk1 was significantly associated with a high grade of differentiation (P=0.0007) and higher rate of distant metastasis (P=0.014). More importantly, decreased levels of Plk1 were associated with absence of response after neoadjuvant therapy (P=0.049). Moreover, low Plk1 expression emerged as an unfavourable prognostic factor for disease-free survival in the non-responder group of patients (P=0.037). CONCLUSIONS Decreased Plk1 expression was associated with poor pathologic response and worse disease-free survival in rectal cancer patients receiving neoadjuvant chemoradiotherapy, suggesting Plk1 as a clinically relevant marker to predict chemoradiotherapy response and outcome.

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.

MicroRNA-31 Emerges as a Predictive Biomarker of Pathological Response and Outcome in Locally Advanced Rectal Cancer

Neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision has emerged as the standard treatment for locally advanced rectal cancer (LARC) patients. However, many cases do not respond to neoadjuvant CRT, suffering unnecessary toxicities and surgery delays. Thus, identification of predictive biomarkers for neoadjuvant CRT is a current clinical need. In the present study, microRNA-31 expression was measured in formalin-fixed paraffin-embedded (FFPE) biopsies from 78 patients diagnosed with LARC who were treated with neoadjuvant CRT. Then, the obtained results were correlated with clinical and pathological characteristics and outcome. High microRNA-31 (miR-31) levels were found overexpressed in 34.2% of cases. Its overexpression significantly predicted poor pathological response (p = 0.018) and worse overall survival (OS) (p = 0.008). The odds ratio for no pathological response among patients with miR-31 overexpression was 0.18 (Confidence Interval = 0.06 to 0.57; p = 0.003). Multivariate analysis corroborated the clinical impact of miR-31 in determining pathological response to neoadjuvant CRT as well as OS. Altogether, miR-31 quantification emerges as a novel valuable clinical tool to predict both pathological response and outcome in LARC patients.

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.