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This Special Issue aims to include relevant works that increase our knowledge about the molecular pathways that govern the development and progression of high-prevalence human cancers, which are responsible for most cancer-related deaths worldwide [...]

Peritoneal metastases from gastric cancer play a key role in the fatal prognosis of the disease. The lack of efficacy of actual therapeutic approaches together with the outcomes achieved with checkpoint inhibitors in gastric cancer compel us to address the current state-of-the-art immunotherapy treatment of peritoneal dissemination. The immunogenicity of the peritoneum has been described to be particularly active at omentum and peritoneal lymph nodes. Also, both innate and acquired immunity seems to be involved at different molecular levels. Recent works show PDL1 expression being less present at the peritoneal level; however, some clinical trials have begun to yield results. For example, the ATTRACTION-2 trial has demonstrated the activity of Nivolumab in heavily pretreated patients even though peritoneal metastases were diagnosed in a 30% of them. Despite positive results in the metastatic setting, peritoneal responses to systemic checkpoint inhibitors remains unclear, therefore, new strategies for intraperitoneal immunotherapy are being proposed for different ongoing clinical trials.

The bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, has emerged in the last years as a promising molecular target in many tumors including breast cancer. The triple negative breast cancer (TNBC) represents the molecular subtype with the worst prognosis and a current therapeutic challenge, and TNBC cells have been reported to show a preferential sensitivity to BET inhibitors. Interestingly, BRD4 phosphorylation (pBRD4) was found as an alteration that confers resistance to BET inhibition and PP2A proposed as the phosphatase responsible to regulate pBRD4 levels. However, the potential clinical significance of pBRD4, as well as its potential correlation with the PP2A pathway in TNBC, remains to be investigated. Here, we evaluated the expression levels of pBRD4 in a series of 132 TNBC patients. We found high pBRD4 levels in 34.1% of cases (45/132), and this alteration was found to be associated with the development of patient recurrences (p = 0.007). Interestingly, BRD4 hyperphosphorylation predicted significantly shorter overall (p &lt, 0.001) and event-free survival (p &lt, 0.001). Moreover, multivariate analyses were performed to confirm its independent prognostic impact in our cohort. In conclusion, our findings show that BRD4 hyperphosphorylation is an alteration associated with PP2A inhibition that defines a subgroup of TNBC patients with unfavorable prognosis, suggesting the potential clinical and therapeutic usefulness of the PP2A/BRD4 axis as a novel molecular target to overcome resistance to treatments based on BRD4 inhibition.

The standard treatment for patients with locally advanced colorectal cancer (LARC) is neoadjuvant 5-fluorouracil (5-FU) based chemoradiotherapy (CRT) followed by surgical mesorec-tal excision. However, the lack of response to this preoperative treatment strongly compromises patient outcomes and leads to surgical delays and undesired toxicities in those non-responder cases. Thus, the identification of effective and robust biomarkers to predict response to preoperative CRT represents an urgent need in the current clinical management of LARC. The oncomiR microRNA-19b (miR-19b) has been reported to functionally play oncogenic roles in colorectal cancer (CRC) cells as well as regulate 5-FU sensitivity and determine outcome in CRC patients. However, its clinical impact in LARC has not been previously investigated. Here, we show that miR-19b deregulation is a common event in this disease, and its decreased expression significantly associates with lower tumor size after CRT (p = 0.003), early pathological stage (p = 0.003), and absence of recurrence (p = 0.001) in LARC patients. Interestingly, low miR-19b expression shows a predictive value of better response to neoajuvant CRT (p < 0.001), and the subgroup of LARC patients with low miR-19b levels have a markedly longer overall (p = 0.003) and event-free survival (p = 0.023). Finally, multivariate analyses determined that miR-19b independently predicts both patient outcome and response to preoperative CRT, highlighting its potential clinical usefulness in the management of LARC patients, This research was funded by PI18/00382 and PI16/01468 grants from “Instituto de Salud Carlos III FEDER”. M.S-A. is supported by “Fundación Cristina Rábago de Jiménez Díaz”

The endogenous PP2A inhibitor SET Nuclear Proto-Oncogene (SET) has been reported to play oncogenic roles and determines poor outcomes in colorectal cancer (CRC). Our group previously showed that miR-199b is deregulated in metastatic CRC, and reduced the cell viability and enhanced the sensitivity of CRC cells to standard induction chemotherapy drugs, mainly through direct negative SET regulation. Clinically, miR-199b downregulation was identified as the molecular mechanism responsible for SET overexpression in around half of metastatic CRC patients. However, the potential clinical value of miR-199b in early-stage CRC remains totally unknown. Thus, here we explored the expression levels of this microRNA in a cohort of 171 early-stage CRC patients using real-time polymerase chain reactions. MiR-199b downregulation was found in 21.6% of cases (37 out of 171) and was significantly associated with those patients with a worse Eastern Cooperative Oncology Group (ECOG) status (p = 0.045). Moreover, miR-199b downregulation predicted shorter overall (p &lt, 0.001) and progression-free survival (p = 0.015). As expected, we next immunohistochemically analyzed SET, observing that it was significantly associated with miR-199b in our cohort. However, multivariate analyses showed that miR-199b was an independent biomarker of poor outcomes in early-stage CRC with a predictive value stronger than SET. In conclusion, our results highlight the potential clinical usefulness of miR-199b and suggest that it could represent a novel molecular target in this disease.

It is well-known that microRNAs (miRNAs) are critical mediators of initiation and disease progression in many human cancers. Rectal cancer is a highly prevalent tumor, accounting for around one third of newly diagnosed colorectal cancers. The usefulness of miRNAs as clinical biomarkers predictive of the outcome and response to chemoradiotherapy has been well-reported for rectal cancer. However, the existing literature on their functional and therapeutic impact needs to be put in context to clarify their role in disease pathogenesis. Therfore, this review is focused on the functional relevance of miRNAs as key regulators of signaling pathways in rectal cancer and their potential therapeutic value as novel molecular targets in this disease., This research was funded by PI18/00382 and PI16/01468 grants from “Instituto de Salud Carlos III FEDER”.

Background Next-generation sequencing (NGS) is a high-throughput technology that has become widely integrated in molecular diagnostics laboratories. Among the large diversity of NGS-based panels, the Trusight Tumor 26 (TsT26) enables the detection of low-frequency variants across 26 genes using the MiSeq platform. Methods We describe the inter-laboratory validation and subsequent clinical application of the panel in 399 patients presenting a range of tumor types, including gastrointestinal (GI, 29%), hematologic (18%), lung (13%), gynecological and breast (8% each), among others. Results The panel is highly accurate with a test sensitivity of 92%, and demonstrated high specificity and positive predictive values (95% and 96%, respectively). Sequencing testing was successful in two-thirds of patients, while the remaining third failed due to unsuccessful quality-control filtering. Most detected variants were observed in the TP53 (28%), KRAS (16%), APC (10%) and PIK3CA (8%) genes. Overall, 372 variants were identified, primarily distributed as missense (81%), stop gain (9%) and frameshift (7%) altered sequences and mostly reported as pathogenic (78%) and variants of uncertain significance (19%). Only 14% of patients received targeted treatment based on the variant determined by the panel. The variants most frequently observed in GI and lung tumors were: KRAS c.35G > A (p.G12D), c.35G > T (p.G12V) and c.34G > T (p.G12C). Conclusions Prior panel validation allowed its use in the laboratory daily practice by providing several relevant and potentially targetable variants across multiple tumors. However, this study is limited by high sample inadequacy rate, raising doubts as to continuity in the clinical setting.

Neoadjuvant 5-fluorouracil (5-FU)-based chemoradiotherapy followed by mesorectal excision is the current standard treatment in locally advanced rectal cancer (LARC) and the lack of complete response represents a major problem that compromises long-term patient survival. However, there is a lack of robust established markers predictive of response to this preoperative treatment available in the clinical routine. The tumor suppressor microRNA (miR)-199b directly targets the PP2A inhibitor SET, which has been involved in 5-FU resistance, and its downregulation has been found to correlate with poor outcome in metastatic colorectal cancer. Here, we studied the functional effects of miR-199b on 5-FU sensitivity after its ectopic modulation, and its expression was quantified by real-time-PCR in a cohort of 110 LARC patients to evaluate its potential clinical significance. Interestingly, our findings demonstrate that miR-199b enhances the sensitivity of colorectal cancer cells to 5-FU in a SET-dependent manner, and that both miR-199b overexpression and SET inhibition are able to overcome resistance to this drug using an acquired 5-FU-resistant model. MiR-199b was found downregulated in 26.4% of cases and was associated with positive lymph node levels after chemoradiotherapy (CRT, p = 0.007) and high pathological stage (p = 0.029). Moreover, miR-199b downregulation determined shorter overall (p = 0.003) and event-free survival (p = 0.005), and was an independent predictor of poor response to preoperative CRT (p = 0.004). In conclusion, our findings highlight the clinical impact of miR-199b downregulation predicting poor outcome and pathological response in LARC, and suggest the miR-199b/SET signaling axis as a novel molecular target to prevent the development of resistance to 5-FU treatment., This research was funded by PI18/00382 and PI16/01468 grants from “Instituto de Salud Carlos III FEDER”

The functional loss of the tumor suppressor protein phosphatase 2A (PP2A) occurs in a wide variety of human cancers including colorectal cancer (CRC), and SET overexpression has been reported as a key contributing mechanism to inhibit PP2A. Although SET binding protein 1 (SETBP1) overexpression and gain of function mutations have been described in several hematological malignancies as common events that increase the expression levels of the PP2A inhibitor SET, thereby leading to PP2A inactivation, the potential existence of SETBP1 alterations in CRC still remains unexplored. We studied the expression profile of SETBP1 by Western blot in a set of CRC cell lines and patient samples. Moreover, we performed co-immunoprecipitation assays to analyze the formation of the previously reported SETBP1–SET–PP2A inhibitory complex. Furthermore, we evaluated the mutational status of SETBP1 by pyrosequencing assays in a cohort of 55 CRC patients with metastatic disease after the immunohistochemical characterization of SET and p-PP2A expression in this cohort. We found high SETBP1 expression in several CRC lines but only in two of the patients analyzed. In addition, we demonstrated the formation of the SETBP1–SET–PP2A heterotrimeric complex in CRC cells. However, we failed to detect SETBP1 mutations in any of the CRC patient samples included in the study. Our results suggest that SETBP1 expression is mainly similar o lower in colorectal cancer tissue compared to normal colonic mucosa. However, its overexpression is a low prevalent alteration which could contribute to inhibit PP2A in CRC through the formation of a SETBP1–SET–PP2A complex in some CRC patients. Moreover, SETBP1 mutations are, if exist, rare events in CRC patients.

The absence of established predictive markers with value to anticipate response to neoadjuvant 5-fluorouracil (5-FU)-based chemoradiotherapy (CRT) represents a current major challenge in locally advanced rectal cancer (LARC). The tumor suppressor microRNA (miR)-199b has been reported to play a key role determining 5-FU sensitivity of colorectal cancer cells through the regulation of several signaling pathways, and has emerged as a novel molecular target to overcome the 5-FU resistant phenotype. Moreover, miR-199b downregulation was described as a common alteration that predicts lack of response to preoperative CRT in LARC but this issue needs to be confirmed in independent larger cohorts. Here, we evaluate the clinical impact of miR-199b in LARC and perform additional analyses to further clarify its potential relevance as novel marker in this disease. Thus, miR-199b expression was quantified by real-time-PCR in a cohort of 185 LARC patients, observing this miR downregulated in 22.2% of cases and significantly associated with higher tumor size (p = 0.026) and positive lymph node after CRT (p = 0.005), and higher pathological stage (p = 0.004). Notably, this alteration showed a strong independent predictive value of poor pathological response to neoadjuvant CRT (p = 0.004). Moreover, the subgroup of cases with low miR-199b levels had a markedly shorter overall (p &lt, 001) and event-free survival (p &lt, 0.001), and multivariate analyses showed that miR-199b deregulation represents an independent prognosticator for patient outcome in LARC. Interestingly, the prognostic impact of this miR was strongly significant in both younger and elderly patients, and was very effective determining patient recurrence (p = 0.004). Finally, we compared miR-199b expression profiles in a set of cases with pre and post-treatment samples available, observing that only a minimal response leads to miR-199b increase levels, further suggesting its potential clinical and therapeutic relevance as a promising marker and novel molecular target for the management of LARC.

Caveolin-1 (CAV-1), the main scaffold protein in caveolae, is frequently deregulated in human cancer. Of importance, this protein has been described to show tumor suppressor or oncogenic properties depending on the cell type and the stage of the disease. In fact, its role in colorectal cancer (CRC) remains to be fully clarified due to discrepancies in the literature. We analyzed CAV-1 by western blot in a set of early-stage CRC patients with paired tumor tissue and normal colonic mucosa available. CAV-1 mRNA and expression levels of miR-124, 133 and 802 were quantified by real-time PCR. We found CAV-1 strongly downregulated in 76.2% of tumor samples and associated with the subgroup of elderly patients (p = 0.027). We observed by real-time PCR a lack of correlation between CAV-1 mRNA and protein levels in some cases with CAV-1 downregulated by western blot, and miR-124 deregulation was identified as a potential contributing alteration to decrease CAV-1 protein expression. CAV-1 is commonly downregulated in patients with primary CRC, which suggests its tumor suppressor role in early stages of this disease. Moreover, based on our findings, the previous discrepancies observed in different studies to date could be due to a complex posttranscriptional CAV-1 regulation.

Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer, leading to improved overall survival. However, acquired resistance inevitably occurs. We aimed to identify, quantify, and assess the mechanisms of acquired resistance to trastuzumab. We established an acquired trastuzumab-resistant model in vitro from BT-474, a trastuzumab-sensitive, HER2-amplified breast-cancer cell line. A multi-omic strategy was implemented to obtain gene, proteome, and phosphoproteome signatures associated with acquired resistance to trastuzumab in HER2-positive breast cancer, followed by validation in human clinical samples. YAP1 dephosphorylation and TEAD2 overexpression were detected as significant alterations in the Hippo pathway in trastuzumab-resistant breast cancer. Because of the emerging role of these proteins as mediators of normal growth and tumorigenesis, we assessed the exogenous modulation of their activity, either by in vitro gene silencing or by pharmacological inhibition of the YAP1/TEAD complexes, both in vitro and in vivo. Moreover, we identified increased signaling through the Hippo pathway in human samples after progression following trastuzumab treatment. Finally, YAP1/TAZ nuclear accumulation in malignant cells in HER2 breast tumor was significantly associated with worse progression-free and overall survival in metastatic HER2-positive breast-cancer patients. Our results suggest the involvement of Hippo signaling in acquired trastuzumab resistance in breast cancer. Additionally, we provide novel evidence for a potential breast-cancer treatment strategy based on dual targeting of HER2 and Hippo pathway effectors, which may improve the antitumor activity of trastuzumab and help overcome resistance., The present work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) with European Regional Development Fund (ERDF) funding through the Institute of Health Carlos III (AES Program, grants PI15/00934, PI18/00382 and PI18/00006; CIBERONC, Biomedical Research Networking Centre for Cancer; Biobanks Platform, PT13/0010/0012; ProteoRed, PRB2-ISCIII, PT13/0001); and the Community of Madrid (S2010/BMD-2344). P.G.-A. was supported by a Fundación Conchita Rábago de Jiménez Díaz grant. P.M. was supported by the ISCIII Miguel Servet Program (CP16/00116).

HER2-positive breast cancer is currently managed with chemotherapy in combination with specific anti-HER2 therapies, including trastuzumab. However, a high percentage of patients with HER2-positive tumors do not respond to trastuzumab (primary resistance) or either recur (acquired resistance), mostly due to molecular alterations in the tumor that are either unknown or undetermined in clinical practice. Those alterations may cause the tumor to be refractory to treatment with trastuzumab, promoting tumor proliferation and metastasis. Using continued exposure of a HER2-positive cell line to trastuzumab, we generated a model of acquired resistance characterized by increased expression of several cytokines. Differential gene expression analysis indicated an overexpression of 15 genes, including five different chemokines, and highlighting CCL5/RANTES as the most overexpressed one. Functional studies, either by in vitro gene silencing or by in vitro and in vivo pharmacologic inhibition of the CCL5/CCR5 interaction with maraviroc, confirmed that CCL5 overexpression was implicated in acquired resistance to trastuzumab, which was mediated by ERK activation. In patient samples, increased CCL5 expression significantly correlated with lower rates of complete response after neoadjuvant therapy, confirmed by detection of high serum CCL5 levels by ELISA. Overexpression of CCL5 correlated with ERK phosphorylation in tumor cells and was statistically associated with worse disease-free survival and overall cancer survival in patients with early HER2-positive breast cancer.

Background: NF-κB signalling appears deregulated in breast tumours. The purpose of this study was to determine whether the non-canonical NF-κB pathway, is activated in oestrogen receptor positive (ER+) breast cancer, to identify any correlation between its activity and the clinico-pathological phenotype and to explore whether NF-κB2 and RelB subunits and/or any of their target genes might be used as a predictive marker. Methods: Two independent cohorts of ER+ early breast cancer patients treated with adjuvant endocrine therapy were included in the study. Activation of RelB and NF-κB2 subunits was determined in a training set of 121 patients by measuring DNA-binding activities in nuclear extracts from fresh frozen specimens by an ELISA-based assay. Samples of 15 ER− breast cancer patients were also included in the study. In a large validation cohort of 207 patients, nuclear immunostaining of RelB and NF-κB2 on formalin-fixed paraffin-embedded specimens was performed. Statistical correlation within clinico-pathological factors, disease-free survival (DFS) and overall survival (OS) was evaluated. Publicly available gene expression and survival data have been interrogated aimed to identify target genes. Results: Activation of NF-κB2 and RelB was found in 53.7 and 49.2% of the 121 ER+ tumours analysed, with similar levels to ER− breast tumours analysed in parallel for comparisons. In the validation cohort, we obtained a similar proportion of cases with activation of NF-κB2 and RelB (59.9 and 32.4%), with a 39.6% of co-activation. Multiplexing immunofluorescence in breast cancer tissue confirmed an inverse spatial distribution of ER with NF-κB2 and RelB nuclear expression in tumour cells. Interestingly, NF-κB2 and RelB mRNA expression was inversely correlated with ER gene (ESR1) levels (P

SET nuclear proto-oncogene (SET) deregulation is a novel molecular target in metastatic colorectal cancer (CRC). However, its role in CRC progression and its potential clinical impact in early-stage CRC patients remain unknown. Here, we studied the biological effects of SET on migration using wound-healing and transwell assays, and anchorage-independent cell growth using soft agar colony formation assays after ectopic SET modulation. SET was analyzed by immuno-staining in 231 early-stage CRC patients, and miR-199b expression was quantified by real-time PCR in a set of CRC patients. Interestingly, SET enhances cell migration, markedly affects the colony-forming ability, promotes epithelial to mesenchymal transition, and induces the expression of the MYC proto-oncogene (c-MYC) in CRC cells. SET overexpression was detected in 15.4% of cases and was associated with worse Eastern Cooperative Oncology Group (ECOG) status (p = 0.021) and relapse in stage-II CRC patients (p = 0.008). Moreover, SET overexpression predicted shorter overall survival (p < 0.001) and time to metastasis (p < 0.001), and its prognostic value was particularly evident in elderly patients. MiR-199b downregulation was identified as a molecular mechanism to deregulate SET in patients with localized disease. In conclusion, SET overexpression is a common alteration in early-stage CRC, playing an oncogenic role associated with progression and aggressiveness, and portends a poor outcome. Thus, SET emerges as a novel potential molecular target with clinical impact in early-stage in CRC.

Background: CSDE1 is located upstream of the N-RAS locus, and codes for an RNA-binding protein named Upstream of N-Ras (UNR). In cancer, CSDE1 has been shown to regulate c-fos, c-myc, pten, rac1 or vimentin. UNR/CSDE1 has been studied in breast, melanoma, pancreatic and prostate cancer. Then, the aim of this study is to evaluate the role of CSDE1/UNR in colorectal cancer progression and maintenance of aggressive phenotype. Methods: For this, we firstly evaluated UNR/CSDE1 expression in human colon cancer derived cell lines, and patient samples. Not only UNR/CSDE1 expression was higher in tumor samples compared to untransformed samples, but also in colonospheres and metastatic origin cell lines than their parental and primary cell lines respectively. Subsequently, we performed functional experiments by UNR/CSDE1 downregulation. Findings: We described for the first time the oncogenic role of UNR/CSDE1 in colorectal cancer. Downregulation of UNR/CSDE1 reduced cell viability and migration throughout a restrain of epithelial-to-mesenchymal transition and increases sensitivity to apoptosis. We then evaluated UNR/CSDE1 expression in two independent set of patients: cohort I with 35 and cohort II with 222 patient samples, and we found that high UNR/CSDE1 expression was associated to poor prognosis. Interestingly, expression of UNR correlated positively with c-MYC expression in colorectal cancer samples and cell lines. Conclusion: Here, we show compelling data reporting the oncogenic role of UNR/CSDE1 in human colorectal cancer samples. Funding: This work has been carried out with the support of the RNA-Reg ConsoliderConsortium (CSD2009-00080), and by Spanish Health Research Project Funds (PI16/01468) from Instituto de Salud Carlos III -FEDER (J.G.-F.), both of the Spanish Ministry of Economy, Industry and Competitiveness. Declaration of Interest: The authors declare no potential conflicts of interest. Ethical Approval: All patients gave written informed consent for the use of their biological samples for research purposes. The institutional review board (IRB) of the Fundacion Jimenez Diaz Hospital evaluated the study, granting approval on December 9, 2014 with approval number 17/14. Moreover, fundamental ethical principles promoted by Spain (LOPD 15/1999) and the European Union Fundamental Rights of the EU (2000/C364/01) were followed. In addition, all patient´s data were processed according to Declaration of Helsinki (last revision 2013) and Spanish National Biomedical Research Law (14/2007, of July 3).

The treatment of choice for locally advanced rectal cancer is preoperative chemoradiotherapy. Despite half of patients do not respond and suffer unnecessary toxicities and surgery delays, there are no biomarkers to guide preoperative CRT outcome. MicroRNA-21 has been related to acquisition of 5-fluorouracil resistance; however, its potential predictive value of response to preoperative chemoradiotherapy in locally advanced rectal cancer remains unknown. Nighty-two patients diagnosed with locally advanced rectal cancer who were preoperatively treated with chemoradiotherapy were selected for this study. Moreover, microRNA-21 expression was quantified in formalin-fixed paraffin-embedded biopsies from this cohort, and the results obtained were correlated with clinical and molecular characteristics, pathological response, and outcome. MicroRNA-21 was found overexpressed in 77.6 % cases, and significantly correlated with tumor grade after preoperative chemoradiotherapy (P = 0.013) and with pathological response (P = 0.013). The odds ratio of having miR-21 overexpression and not getting a respond to chemoradiotherapy resulted in 9.75 CI 2.24 to 42. Sensitivity, specificity, negative predictive values, and positive predictive value were 86.6, 60, 42.8, and 92 %, respectively. Multivariate analysis confirmed the clinical significance of miR-21 determining preoperative chemoradiotherapy response. MicroRNA-21 expression efficiently predicts preoperative chemoradiotherapy pathological response in locally advanced rectal 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.

Background: Sunitinib represents a widely used therapy for metastatic renal cell carcinoma patients. Even so, there is a group of patients who show toxicity without clinical benefit. In this work, we have analysed pivotal molecular targets involved in angiogenesis (vascular endothelial growth factor (VEGF)-A, VEGF receptor 2 (KDR), phosphorylated (p)KDR and microvascular density (MVD)) to test their potential value as predictive biomarkers of clinical benefit in sunitinib-treated renal cell carcinoma patients. Methods: Vascular endothelial growth factor-A, KDR and pKDR-Y1775 expression as well as CD31, for MVD visualisation, were determined by immunohistochemistry in 48 renal cell carcinoma patients, including 23 metastatic cases treated with sunitinib. Threshold was defined for each biomarker, and univariate and multivariate analyses for progression-free survival (PFS) and overall survival (OS) were carried out. Results: The HistoScore mean value obtained for VEGF-A was 121.6 (range, 10–300); for KDR 258.5 (range, 150–300); for pKDR-Y1775 10.8 (range, 0–65) and the mean value of CD31-positive structures for MVD visualisation was 49 (range, 10–126). Statistical differences for PFS (P=0.01) and OS (P=0.007) were observed for pKDR-Y1775 in sunitinib-treated patients. Importantly, pKDR-Y1775 expression remained significant after multivariate Cox analysis for PFS (P=0.01; HR: 5.35, 95% CI, 1.49–19.13) and for OS (P=0.02; HR: 5.13, 95% CI, 1.25–21.05). Conclusions: Our results suggest that the expression of phosphorylated (i.e., activated) KDR in tumour stroma might be used as predictive biomarker for the clinical outcome in renal cell carcinoma first-line sunitinib-treated patients.

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.

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.

Dear Editor, We have read with great interest the recent published manuscript by Caiazza et al. (2016), which provides novel exciting findings about the potential therapeutic value of enzalutamide in patients with androgen receptor (AR)-positive triple negative breast cancer (TNBC). Some previous studies have shown promising antitumor effects of this drug in breast tumors. Thus, enzalutamide-mediated AR inhibition has been reported to reduce proliferation, anchorage-independent growth, migration, and invasion and increases apoptosis of TNBC cells in vitro and decrease viability of TNBC xenografts in vivo (Cochrane et al. 2014; Barton et al. 2015). In concordance with these data, Caiazza and co-workers found that enzalutamide reduced cell growth and clonogenic potential as well as migration and invasion capabilities of breast cancer cells. Moreover, the authors showed that enzalutamide exerts its antitumor effects through an AR-dependent manner and probably modulating the expression of the transcription factors AP-1 and SP-1. Of note, it has been recently described that targeting the PP2A/SET signalling axis is able to overcome the enzalutamide resistance in castration-resistant prostate cancer cells (Hu et al. 2015). Interestingly, PP2A is a largely reported tumor suppressor frequently inactivated in human cancer that regulates both AP-1 and SP-1 activation as well as AR expression (Al-Murrani et al. 1999; Huang et al. 2009). Our group has reported that PP2A is inhibited in breast cancer, with a particularly higher incidence in TNBC. In addition, this alteration represents a novel therapeutic target that defines a subgroup of breast cancer patients with very poor outcome (Rincon et al. 2015). Altogether, these data highlight the potential therapeutic value of targeting PP2A to prevent or overcome enzalutamide resistance in AR+ TNBC. Furthermore, AR is known to be expressed in around 90% of estrogen receptor (ER)-positive breast tumors. The fact that AR is required for maximum ER genomic binding and activity determines the synergistic effect observed when anti-androgens such as enzalutamide are used in combination with traditional endocrine therapy with tamoxifen or fulvestrant. In fact, enzalutamide has been found effective even as a single agent in tumors resistant to these treatments (Cochrane et al. 2014; D´Amato et al. 2016). These findings are concordant with the work by Caiazza and co-workers, showing that antitumor effects of enzalutamide were similar in TN and non-TN cell lines but dependent on the AR expression. In this line of thinking, PP2A has been also found to directly regulate ER expression (Lu et al. 2003) and its pharmacologic reactivation using FTY720 serves to enhance sensitivity of breast cancer cells to tamoxifen (Hait et al. 2015). Altogether, these data would support the potential benefit derived from the use of enzatulamide in combination with PP2A activating drugs also in the set of AR+/ER+ breast cancer tumors. In conclusion, the potential clinical usefulness of this novel therapeutic strategy based on the inclusion of enzalutamide in combination with PP2A activators for treating AR+ breast cancers should be further confirmed in forthcoming studies.

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., This work was supported by PT13/0010/0012, PI13/02609 and PI15/00934 grants from “Instituto de Salud Carlos III FEDER, Madrid (Spain)”

Background: Overexpression and amplification of the human epidermal growth factor receptor 2 (HER2) occur in 20% of total breast carcinomas. HER2-overexpression is implicated in disease initiation and progression and associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody, is the standard HER2-targeted therapy for early and metastatic HER2-amplified breast cancer patients. Trastuzumab has significantly increased clinical benefit in HER2+ metastatic and adjuvant settings; however, it is not effective for many patients due to primary or acquired resistance to the drug. During the last decade, many studies have revealed a number of novel molecular traits of HER2+ breast cancer, allowing us to uncover the molecular mechanisms involved in trastuzumab resistance and develop strategies to overcome resistance to therapy. Objective: In this review, we comprehensively addressed the current achievements in preclinical studies; we discussed molecular mechanisms of acquired trastuzumab resistance in HER2+ breast cancer models and potential therapeutic approaches based on the molecular features for HER2+ breast cancer. Conclusion: Enhanced understanding of the molecular profiles in HER2+ breast cancer may lead to the identification of novel biomarkers for the development of diagnostic approaches and improvement of therapeutic targets for the prevention and treatment of trastuzumab resistant HER2+ breast cancer.

Hepatoblastoma is the most common hepatic malignancy during childhood. However, little is still known about the molecular mechanisms that govern the development of this disease. This review is focused on the recent advances regarding the study of microRNAs in hepatoblastoma and their substantial contribution to improv our knowledge of the pathogenesis of this disease. We show here that miRNAs represent valuable tools to identify signaling pathways involved in hepatoblastoma progression as well as useful biomarkers and novel molecular targets to develop alternative therapeutic strategies in this disease, This research was funded by PI15/00934 and PI16/01468 grants from Instituto de Salud Carlos III FEDER.

FTY720 (Fingolimod, Gilenya (†) ) is an FDA-approved immunosuppressant currently used in the treatment of multiple sclerosis. However, a large number of studies over the last few years have shown that FTY720 shows potent antitumor properties that suggest its potential usefulness as a novel anticancer agent. Interestingly, the restoration of protein phosphatase 2A (PP2A) activity mediated by FTY720 could play a key role in its antitumor effects. Taking into account that PP2A inactivation is a common event that determines poor outcome in several tumor types, FTY720 could serve as an alternative therapeutic strategy for cancer patients with such alterations.

// Ion Cristobal 1, * , Cristina Carames 1, * , Raul Rincon 1 , Rebeca Manso 2 , Juan Madoz-Gurpide 2 , Blanca Torrejon 1 , Paula Gonzalez-Alonso 2 , Federico Rojo 2 and Jesus Garcia-Foncillas 1 1 Translational Oncology Division, Oncohealth Institute, IIS-Fundacion Jimenez Diaz, UAM, University Hospital “Fundacion Jimenez Diaz”, E-28040 Madrid, Spain 2 Pathology Department, University Hospital “Fundacion Jimenez Diaz”, Autonomous University of Madrid, E-28040 Madrid, Spain * These authors contributed equally to this work Correspondence to: Ion Cristobal , email: ion.cristobal@fjd.es Jesus Garcia-Foncillas, email: jgfoncillas@gmail.com Federico Rojo, email: frojo@fjd.es Keywords: miR-199b, SET, PP2A, prognosis, therapy Received: November 02, 2015 Accepted: July 17, 2016 Published: August 10, 2016 ABSTRACT 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.

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., The current work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; AES Program, grants PI12/01552; PI12/00680; PI12/01421); the Ministry of Health (Cancer Network); the Community of Madrid (S2010/BMD-2344 grant); and the Government of Catalonia (2014/SGR/740 grant). The biobanks are funded by grants from the MINECO (Institute of Health Carlos III, RETICS Biobanks Network, with FEDER funds: Fundación Jiménez Díaz Biobank, RD12/0036/0021; Parc de Salut Mar Biobank, RD12/0036/0051; Valencia Clinic Hospital Biobank, RD12/0036/0070). S. Zazo and C. Chamizo are supported by grants from the Biobanks initiative. J. Albanell and F. Rojo are recipients of an intensification program ISCIII/FEDER. R. Manso and P. González-Alonso are supported by Fundación Conchita Rábago de Jiménez Díaz grants.

Background: Activation of the MET oncogene promotes tumor growth, invasion and metastasis in several tumor types. Additionally, MET is activated as a compensatory pathway in the presence of EGFR blockade, thus resulting in a mechanism of resistance to EGFR inhibitors. Methods: We have investigated the impact of HGF and MET expression, MET activation (phosphorylation), MET gene status, and MET-activating mutations on cetuximab sensitivity in recurrent or metastatic squamous cell carcinoma of the head and neck (HNSCC) patients. Results: A single-institution retrospective analysis was performed in 57 patients. MET overexpression was detected in 58 % patients, MET amplification in 39 % and MET activation (p-MET) in 30 %. Amplification was associated with MET overexpression. Log-rank testing showed significantly worse outcomes in recurrent/metastatic, MET overexpressing patients for progression-free survival and overall survival. Activation of MET was correlated with worse PFS and OS. In multivariate logistic regression analysis, p-MET was an independent prognostic factor for PFS. HGF overexpression was observed in 58 % patients and was associated with MET phosphorylation, suggesting a paracrine activation of the receptor. Conclusions: HGF/MET pathway activation correlated with worse outcome in recurrent/metastatic HNSCC patients. When treated with a cetuximab-based regimen, these patients correlated with worse outcome. This supports a dual blocking strategy of HGF/MET and EGFR pathways for the treatment of patients with recurrent/metastatic HNSCC, The present work was supported by grants from the Spanish Ministerio de Economia y Competitividad (MINECO) (AES Program, grant PI12/01552); the Ministerio de Sanidad (Cancer Network); the Comunidad de Madrid (S2010/BMD-2344). The Fundacion Jimenez Diaz Biobank is funded by a grant from the MINECO (Instituto de Salud Carlos III, RETICS Red de Biobancos, with FEDER funds, RD09/0076/00101). S.Z. and C.C. are supported by grants from the same Biobanks initiative

Background: Although it has been suggested that a high level of thymidylate synthase (TYMS) gene expression in malignant tumors is related to reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in routine clinical samples from patients treated with the drug. The purpose of this study was to quantitatively assess the impact of TYMS gene expression in tumor cells as a predictor of the efficacy of pemetrexed therapy in patients with advanced non-small cell lung cancer (NSCLC) treated at our institution. Methods: Sixty-two NSCLC patients were included in this study: 16 patients received platins-pemetrexed as first-line NSCLC, and 46 pemetrexed in monotherapy as second- or subsequent-line treatment. Total mRNA was isolated and the expression of TYMS was analyzed by RT-qPCR. TYMS levels were calibrated against expression in normal lung tissue. Results: TYMS overexpression was detected in 61 % of patients and low expression in 39 %. The response rate for patients with low TYMS expression was 0.29 compared with 0.03 in patients with overexpression (P = 0.025). A significant benefit was observed in patients with low expression both in time to progression (average TTP = 56 vs. 23 months, P = 0.001) and in overall survival (average OS = 60 vs. 25 months, P = 0.002). Conclusions: TYMS overexpression in tumor cells correlated with a reduced response to pemetrexed-containing chemotherapy and might be used as a predictive biomarker in advanced NSCLC patients, The present work was supported by grants from the Spanish Ministerio de Economia y Competitividad (MINECO) (AES Program, grant PI12/01552); the Ministerio de Sanidad (Cancer Network); and the Comunidad de Madrid (S2010/BMD-2344). The Fundacion Jimenez Diaz Biobank is funded by a grant from the MINECO (Instituto de Salud Carlos III, RETICS Red de Biobancos, with FEDER funds, RD09/0076/00101). S.Z. and C.C. are supported by grants from the same Biobanks initiative

e12007 Background: Breast cancer prognosis is influenced by several factors including Ki67 expression which may have a predictive role in luminal breast cancer patients. The aim of this study was to assess the value of Ki67 in breast cancers with positive hormonal receptors and HER2 overexpressed and to evaluate its impact on survival. Methods: Seventy eight consecutive patients diagnosed with locally advanced HER2 positive breast cancer who were treated with adjuvant therapy based on HER2 treatment were selected for this study (2004-2014). The adjuvant chemotherapy schemes were 44% TCH, 16% FEC, 11% AC/T and 29% other therapies. The median of followup was 68 months. Tumor proliferation was assessed immunohistochemically by Ki67 expression and calculated as percentage of stained tumor cells from this cohort previous to adjuvant chemotherapy administration and the results obtained were correlated with disease status and outcome. Results: High proliferation defined as a percentage > 15 of tumor cells was observed in 69% of the breast cancer patients cases. High proliferation significantly predicted longer overall survival (OS) (Log rank 0,012). At 10 years of follow-up, 93% of the patients with KI67 high expression were alive versus 43% of patients without overexpression. Multivariate analysis confirmed the clinical significance of Ki67 predicting OS for luminal HER2 breast cancer patients (p 0,04 y HR 9,6). Conclusions: High proliferation identifies a setting of luminal-B HER2+ subtype breast cancer patients with a significantly longer overall survival.

We have read with interest the recent article by Zhang et al. [1], which provides novel exciting findings about the role of miR29c in colorectal cancer (CRC) progression and metastasis. Although little is known about the significance of this microRNA in CRC, some previous studies suggest its potential tumor suppressor role in this disease. Thus, miR-29c has been reported to be expressed at low levels in CRC [2, 3] and also to be predictive of CRC early recurrence [3]. In concordance with these data, the authors found that miR-29c was markedly downregulated in primary CRC tissues from patients with distant metastasis, and predicted worse outcome. Interestingly, miR-29c showed higher expression in liver metastatic tissues in comparison with primary tumor specimens. This observation suggests that miR29c downregulation could be a transient relevant event in CRC progression to metastatic disease. Moreover, miR-29c reduced epithelial-to-mesenchymal transition, cell migration and invasion abilities of CRC cells, and metastasis development in vivo, further supporting its role in CRC metastatic development [1]. However, the precise status of miR-29c in metastatic CRC tissues is a major question to be fully investigated. Thus, we quantified miR-29c in primary and paired metastatic tissues from 17 CRC patients, 12 with liver metastasis and 5 with lung metastasis, using Taqman Low Density Arrays (TLDAs) panel A (Applied Biosystems). A pathologist reviewed the specimens to further confirm the diagnosis. All samples were taken anonymously and the ethical committee and institutional review board approved the project. Relative gene expression analysis was carried out using the 2 method and U6B as internal control. We observed similar miR-29c expression between primary and metastatic tissues from those 12 CRC patients with liver metastasis. However, almost threefold increased miR-29c levels were found in the metastatic tissues of those cases with lung metastasis. We then investigated whether these differences were due to expression changes in the primary tissues or dependent on the metastatic site. Interestingly, miR-29c showed significantly lower expression in primary CRC tissues from patients with lung metastasis compared with those cases with liver metastasis (P = 0.046). No differences were observed when comparing miR-29c between liver and lung CRC metastatic tissues (P = 0.934). In summary, our results would indicate that deeper miR-29c downregulation would be required in the premetastatic CRC cell to develop lung metastasis than liver metastasis. In addition, the potential predictive value of miR-29c determining the CRC metastatic niche should be further confirmed in forthcoming studies.

Background: MicroRNA-214 (miR-214) has been shown to act as a tumour suppressor in human cervical and colorectal cancer cells. The aim of this study was to experimentally validate high mobility group AT-hook 1 as a novel target for miR-214-mediated suppression of growth and motility. Methods: HMGA1 and miR-214 expression levels were estimated in cervical and colorectal clinical specimens using qPCR. HMGA1 3′ untranslated region luciferase assays were performed to validate HMGA1 as a target of miR-214. Effect of altering the expression of miR-214 or HMGA1 on proliferation, migration and invasion of human cervical and colorectal cancer cells was investigated. Results: miR-214 expression was poor while that of HMGA1 was high in cervical and colorectal cancer tissues. miR-214-re-expression or HMGA1 downregulation inhibited proliferation, migration and invasion of cancer cells while miR-214 inhibition had opposite effects. miR-214 was demonstrated to bind to the wild-type 3′ untranslated region of HMGA1 but not with its mutant. Conclusions: Low expression of miR-214 concurrent with elevated levels of HMGA1 may contribute to cervical and colorectal cancer progression. miR-214-mediated regulation of HMGA1 is a novel mechanism for its tumour-suppressive actions in human cervical and colorectal cancer cells and opens up avenues for novel therapeutic strategies for these two cancers.

We have read with great interest the recent work by Ross et al ,1 which provides novel relevant findings about the therapeutic efficacy of using protein phosphatase 2A (PP2A)-activating drugs to target tristetraprolin (TTP) in rheumatoid arthritis (RA). In this elegant work, the authors showed that TTP is overexpressed and colocalises with activated mitogen-activated protein kinase (MAPK) p38 in RA synovial tissue. MAPK p38 phosphorylates and inhibits TTP at two serine residues, and Ross et al determined that these phosphorylation sites are critical for …

Article first published online 16 June 2015. Supplemental Digital Content is Available in the Text., Background: Longstanding ulcerative colitis (UC) bears a high risk for development of UC-associated colorectal carcinoma (UCC). The inflammatory microenvironment influences microRNA expression, which in turn deregulates target gene expression. microRNA-26b (miR-26b) was shown to be instrumental in normal tissue growth and differentiation. Thus, we aimed to investigate the impact of miR-26b in inflammation-associated colorectal carcinogenesis. Methods: Two different cohorts of patients were investigated. In the retrospective group, a tissue microarray with 38 samples from 17 UC/UCC patients was used for miR-26b in situ hybridization and quantitative reverse transcription polymerase chain reaction analyses. In the prospective group, we investigated miR-26b expression in 25 fresh–frozen colon biopsies and corresponding serum samples of 6 UC and 15 non-UC patients, respectively. In silico analysis, Ago2-RNA immunoprecipitation, luciferase reporter assay, quantitative reverse transcription polymerase chain reaction examination, and miR-26b mimic overexpression were employed for target validation. Results: miR-26b expression was shown to be upregulated with disease progression in tissues and serum of UC and UCC patients. Using miR-26b and Ki-67 expression levels, an UCC was predicted with high accuracy. We identified 4 novel miR-26b targets (DIP1, MDM2, CREBBP, BRCA1). Among them, the downregulation of the E3 ubiquitin ligase DIP1 was closely related to death-associated protein kinase stabilization along the normal mucosa-UC-UCC sequence. In silico functional pathway analysis revealed that the common cellular pathways affected by miR-26b are highly related to cancerogenesis and the development of gastrointestinal diseases. Conclusions: We suggest that miR-26b could serve as a biomarker for inflammation-associated processes in the gastrointestinal system. Because miR-26b expression is downregulated in sporadic colon cancer, it could discriminate between UCC and the sporadic cancer type.

We have read with great interest the recently published work by Lin et al (2014), which provides novel relevant findings about the tumour suppressor role of TAp63 via miR-133b downregulation in colorectal cancer (CRC). Of importance, the authors identified miR-133b as a transcriptional target of TAp63, and showed that the modulation of miR-133b expression is essential for the inhibitory effects of TAp63 in CRC cell migration and invasion. Moreover, they showed that TAp63 is expressed at low levels in CRC and proposed this alteration as a potential cause of miR-133b downregulation, which was previously described by our group in CRC cell lines and patient samples (Bandres et al, 2006). Furthermore, it has been reported that miR-133b has a tumour suppressor role inhibiting cell growth through modulation of the MET signalling pathway (Hu et al, 2010), and it has also been described that low expression level of miR-133b correlates with poor clinical outcome in CRC (Akcakaya et al, 2011). Notably, although the findings provided by Lin et al (2014) highlight the potential relevance of miR-133b deregulation in CRC progression and metastasis, this issue needs to be fully clarified. A recent publication pointed out that miR-133b contributes to increased CRC cell migration and invasion, and identified CXCR4 as a direct miR-133b target. In that work, Duan et al (2014) analysed 31 CRC patients observing miR-133b downregulation in 29 out of 31 tumour samples, and much lower expression in metastatic tumours. The authors proposed that miR-133b could be having a relevant role in CRC invasion and metastasis. However, only 13 out of the 31 CRC patients had metastatic disease (9 with lymph node metastasis and 4 with liver metastasis). Therefore, further studies confirming the role of miR-133b in the metastatic cohort are warranted. In this line of thinking, we analysed the potential role of miR-133b in CRC progression and metastasis. We quantified the expression pattern of 377 mature microRNAs using Taqman Low Density Arrays (TLDAs) panel A (Applied Biosystems, Grand Island, NY, USA) in primary and paired metastatic tissues from 17 CRC patients, 12 with liver metastasis and 5 with lung metastasis previously reviewed by a pathologist (FR) 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 2−ΔCt method and U6B was used as internal control. Downregulation was considered when expression in the metastatic tissue showed at least three-fold decrease compared with its paired primary CRC tissue. Interestingly, we found miR-133b significantly downregulated in liver metastatic tissues compared with their paired primary CRC tissues (P