Juan Madoz-Gúrpide

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.

Activation of the Tumor Suppressor PP2A Emerges as a Potential Therapeutic Strategy for Treating Prostate Cancer

Protein phosphatase 2A (PP2A) is a tumor suppressor complex that has recently been reported as a novel and highly relevant molecular target in prostate cancer (PCa). However, its potential therapeutic value remains to be fully clarified. We treated PC-3 and LNCaP cell lines with the PP2A activators forskolin and FTY720 alone or combined with the PP2A inhibitor okadaic acid. We examined PP2A activity, cell growth, prostasphere formation, levels of PP2A phosphorylation, CIP2A and SET expression, and AKT and ERK activation. Interestingly, both forskolin and FTY720 dephosphorylated and activated PP2A, impairing proliferation and prostasphere formation and inducing changes in AKT and ERK phosphorylation. Moreover, FTY720 led to reduced CIP2A levels. Treatment with okadaic acid impaired PP2A activation thus demonstrating the antitumoral PP2A-dependent mechanism of action of both forskolin and FTY720. Levels of PP2A phosphorylation together with SET and CIP2A protein expression were studied in 24 PCa patients and both were associated with high Gleason scores and presence of metastatic disease. Altogether, our results suggest that PP2A inhibition could be involved in PCa progression, and the use of PP2A-activating drugs might represent a novel alternative therapeutic strategy for treating PCa patients.

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

PP2A inhibition as a novel therapeutic target in castration-resistant prostate cancer.

Protein phosphatase 2A (PP2A) is a well-known tumor suppressor frequently inhibited in human cancer. Alterations affecting PP2A subunits together with the deregulation of endogenous PP2A inhibitors such as CIP2A and SET have been described as contributing mechanisms to inactivate PP2A in prostate cancer. Moreover, recent findings highlight that functional inactivation of PP2A could represent a key event in the acquisition of castration-resistant phenotype and a novel molecular target with high impact at both clinical and therapeutic levels in prostate cancer.

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.

Cross Talk between Wnt/β-Catenin and CIP2A/Plk1 Signaling in Prostate Cancer: Promising Therapeutic Implications

ABSTRACT Aberrant activation of the Wnt/β-catenin pathway and polo-like kinase 1 (Plk1) overexpression represent two common events in prostate cancer with relevant functional implications. This minireview analyzes their potential therapeutic significance in prostate cancer based on their role as androgen receptor (AR) signaling regulators and the pivotal role of the tumor suppressor protein phosphatase 2A (PP2A) modulating these pathways.

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.

Potential therapeutic value of miR-425-5p in metastatic colorectal cancer.

The recently published manuscript by Zhang et al. [1] provides novel important data about the role of miR-425-5p modulating chemosensitivity of colorectal cancer (CRC) cells to 5-fluorouracil (5-FU) and oxaliplatin treatments. The authors carried out a comparison between microRNA expression profiles from chemosensitive and chemoresistant HCT-116 cells, and miR-425-5p was found markedly overexpressed in chemoresistant HCT-116 cells. Interestingly, miR-425-5p inhibition had no effects on cell growth and apoptosis in untreated cells but restored chemosensitivity in HCT-116 resistant cells both in vitro and in vivo. These observations are of high therapeutic relevance since, as properly indicated by Zhang et al., 5-FU and oxaliplatin are currently used as standard induction chemotherapy in metastatic CRC but, unfortunately, many patients develop chemoresistance during the disease progression. However, some issues should be taken into consideration. One criticism would be that those experiments showing enhanced chemosensitivity after miR-425-5p inhibition should have been done with 5-FU in combination with oxaliplatin to mimic the FOLFOX regimen rather than separately. On the other hand, the development of HCT-116 resistant cells is one key point in this study and obtaining up to 20-fold increase in 5-FU/oxaliplatin IC50 values is unusual when a resistant cell line is generated by a continuous exposure to these drugs. Thus, it would be very useful for reproducibility if the authors would have provided additional information at this regard such as how much time was needed to achieve this high chemoresistance. In concordance with this observation, it is also surprising that neither 5-FU nor oxaliplatin were able to induce any effect in xenograft models injected with HCT-116 resistant cells. One would expect that a combination of these agents could induce at least some modest tumour growth inhibition. Furthermore, the authors demonstrated that PDCD10 is a novel direct target of miR-425-5p and claimed that the effects of miR-4255p regulating chemosensitivity in CRC cells are dependent on PDCD10 modulation. However, it remains necessary to analyse the potential contribution of other described targets for this microRNA such as the tumour suppressor Phosphatase and tensin homologue (PTEN) [2]. In fact, high PTEN expression has been reported to be associated with sensitivity to 5-FU and oxaliplatin in CRC [3]. Although little is known about the functional significance of miR425-5p, some data in the literature suggest that this microRNA could play a potential oncogenic role in cancer progression. As instance, it has been recently reported that miR-425-5p contributes to invasion and metastasis development in gastric cancer [4]. We then investigated the status of miRNA-425-5p in CRC progression by Taqman low density arrays panel A (Applied Biosystems, Foster City, CA, USA) in 17 CRC patients with paired primary and metastatic tissue available. All samples were anonymized and de-identified prior to analysis and the ethical committee and institutional review board approved the project. Relative gene expression analysis was performed with the 2 DCt method and U6B as internal control. Interestingly, we found no differences in miR-425-5p between primary and metastatic tissues (fold change = 0.94; P = 0.842). These results would suggest that miR-425-5p deregulation occurs early in the primary tumour and its expression levels prior to metastatic progression may predict future response to chemotherapy. However, it seems not to be critical in the progression to metastatic disease. As a result of the short number of cases analysed confirmation in additional studies including larger cohorts is required. In conclusion, the study by Zhang et al. provides novel important findings about the ability of miR-425-5p to regulate chemoresistance in CRC but the fact that their results were obtained only in HCT-116 cells represents a major limitation of this study. Therefore, further investigation in additional CRC models with distinct PTEN status and PDCD10 expression are warranted to clarify the role of these two proteins in this issue and confirm miR-4255p as a novel reliable therapeutic target in CRC. Moreover, it would be very interesting to assess in forthcoming studies the potential value of miR-425-5p deregulation as a predictive marker of response to FOLFOX regimen together with the contribution to chemoresistance of some other deregulated microRNAs identified by Zhang and colleagues in their work.