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Vitamin C uncouples the Warburg metabolic switch in KRAS mutant colon cancer

KRAS mutation is often present in many hard-to-treat tumors such as colon and pancreatic cancer and it is tightly linked to serious alterations in the normal cell metabolism and clinical resistance to chemotherapy. In 1931, the winner of the Nobel Prize in Medicine, Otto Warburg, stated that cancer was primarily caused by altered metabolism interfering with energy processing in the normal cell. Increased cell glycolytic rates even in the presence of oxygen is fully recognized as a hallmark in cancer and known as the Warburg effect. In the late 1970′s, Linus Pauling and Ewan Cameron reported that vitamin C may have positive effects in cancer treatment, although deep mechanistic knowledge about this activity is still scarce. We describe a novel antitumoral mechanism of vitamin C in KRAS mutant colorectal cancer that involves the Warburg metabolic disruption through downregulation of key metabolic checkpoints in KRAS mutant cancer cells and tumors without killing human immortalized colonocytes. Vitamin C induces RAS detachment from the cell membrane inhibiting ERK 1/2 and PKM2 phosphorylation. As a consequence of this activity, strong downregulation of the glucose transporter (GLUT-1) and pyruvate kinase M2 (PKM2)-PTB dependent protein expression are observed causing a major blockage of the Warburg effect and therefore energetic stress. We propose a combination of conventional chemotherapy with metabolic strategies, including vitamin C and/or other molecules targeting pivotal key players involved in the Warburg effect which may constitute a new horizon in anti-cancer therapies.

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.

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.

Downregulation of miR-138 as a Contributing Mechanism to Lcn-2 Overexpression in Colorectal Cancer with Liver Metastasis

We have read the interesting manuscript by Maier et al. [1], which analyzes the clinical impact of lipocalin-2 (Lcn-2) in colorectal cancer (CRC). Higher Lpc-2 expression levels were observed in CRC samples compared to normal colonic mucosa confirming data published by others [2]. Moreover, the authors found Lpc-2 overexpression significantly associated with metastasis development, and determined worse overall survival (OS) and disease-free survival (DFS) in CRC patients. There was controversy about the prognostic value of Lcn-2 in CRC since contradictory results has been reported to date. Thus, the results of the study by Maier and co-workers serve to clarify this issue and highlight the role of Lcn-2 in tumor progression together with its prognostic value in CRC. Furthermore, high serum levels of Lcn-2 has been described to correlate with shorter disease-free survival and higher neoplastic tissue volume in metastatic CRC patients further supporting the role of Lcn-2 in CRC tumor progression and malignant development [3]. One major question that still remains to be clarified is the Lcn-2 molecular regulatory mechanisms. Interestingly, miR-138 has been reported to target Lcn-2 then decreasing cell migration and tumor groth in vivo [4]. Moreover, miR138 is low expressed in both CRC tissues and cell lines, and its downregulation is associated with lymph node and distant metastasis development [5]. However, the precise status of miR-138 in metastatic CRC tissues remains to be fully investigated. These observations prompted us to analyze the importance of miR-138 deregulation as a contributing mechanism to Lcn-2 overexpression in metastatic CRC. Thus, we quantified miR-138 in 17 CRC patients with primary and paired metastatic tissues using Taqman low density arrays (TLDAs) panel A (Applied Biosystems). The specimens included in this work were previously reviewed by a pathologist 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 performed using the 2 Dct method and U6B as internal control. As expected, we found that more than two-fold decreased miR-138 levels in metastatic CRC tissues compared with primary CRC tissues, although differences did not achieve significance (P = 0.107). We next stratified our set of cases by metastatic site, observing that miR-138 was markedly downregulated in the metastatic tissues of those 12 CRC patients with liver metastasis (P = 0.010). However, we found no differences between primary and metastatic tissues from those cases with lung metastasis (P = 0.194). In conclusion, our results would indicate that & Ion Cristóbal ion.cristobal@fjd.es

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.

Comment on ‘MicroRNA-199b-5p attenuates TGF-β1-induced epithelial–mesenchymal transition in hepatocellular carcinoma’

We read with great interest the recent publication by Zhou and colleagues, which provides novel relevant data about the role of microRNA-199b-5p (miR-199b) and N-cadherin as regulators of epithelial–mesenchymal transition (EMT) in human hepatocellular carcinoma (HCC). In this work, the authors demonstrate that N-cadherin is a direct miR-199b target, and observed that miR-199b downregulation in HCC patients predicts poor outcome and inversely correlates with N-cadherin overexpression. At the functional level, they demonstrated that miR-199b inhibits both migratory and invasive abilities of HCC cells, and metastatic progression in vivo. In addition, they identified a positive regulatory loop between N-cadherin, TGF-β1, and Akt, and showed that miR-199b reduces TGF-β1-induced Akt phosphorylation, thereby affecting EMT in HCC. Despite that this is a very interesting piece of work that provides novel important findings, some considerations should be taken into account. From our point of view, it would be very important to evaluate the potential relevance of alternative miR-199b targets and even more strongly in the work by Zhou et al. due to the following rationale. The authors showed in their work that the functional effects of miR-199b were only partially reversed by N-cadherin restoration, which highlights the involvement of additional molecular targets. Of note, miR-199b has been reported to target SET, which has been recently confirmed by our group in colorectal cancer, showing that SET is of key relevance for miR199b-induced effects. Interestingly, SET has been reported to have a relevant role in EMT in pancreatic cancer via N-cadherin regulation. SET overexpression was found to activate signaling through Rac1/JNK/cJun, thereby inducing the transcriptional activation of N-cadherin expression. In fact, SET overexpression positively correlated with N-Cadherin levels. These observations would indicate that the direct miR-199b-induced N-cadherin regulation would be indirectly reinforced by modulating SET. Furthermore, the authors showed that miR-199b predicted poor outcome in their HCC cohort and reduced TGF-β1-induced Akt phosphorylation. In concordance, SET overexpression has been described to confer poor clinical outcome and correlate with p-Akt expression in HCC tumors. SET is a potent endogenous inhibitor of the tumor suppressor protein phosphatase 2A (PP2A), and PP2A activation has been reported to suppress TGF-β1-induced EMT. In addition, PP2A directly targets and dephosphorylates Akt, which has also been described in HCC. In fact, SET targeting using an antagonist to this protein restored PP2A-mediated p-Akt downregulation and induced apoptosis of HCC cells. Finally, Zhou et al. found that miR-199b inhibits both migration and invasion in HCC cells, and some evidence highlights the potential involvement of SET in these effects. First, SET has a role inducing Rac1-mediated migration. Second, a recent work has described that matrix metalloproteinase-9 (MMP-9) deregulation through PP2A inhibition is a relevant event in HCC that contributes to the tumor invasion, and the PP2A inhibitor SET has been reported to induce MMP-9 expression activity in breast and nonsmall cell lung cancer. Altogether, the work by Zhou et al. suggest that miR-199b/N-cadherin deregulation is a relevant event that predicts poor outcome and contributes to EMT in HCC. However, forthcoming studies are warranted to confirm the wide range of evidence indicating that SET could also be having a key role in the miR-199b/N-cadherin interplay.

PP2A regulates signaling through hormonal receptors in breast cancer with important therapeutic implications

The functional inhibition of protein phosphatase 2A (PP2A) has emerged in the last years as a common alteration in breast cancer that determines poor outcome and contributes to disease progression and aggressiveness. Furthermore, expression of estrogen receptor (ER) is a high relevant molecular event with key therapeutic implications in breast cancer, and androgen receptor (AR) signaling is involved in the pathogenesis of breast cancer and represents a novel target with crescent importance in this disease. In this review, we summarize the role of the tumor suppressor PP2A in modulating ER and AR signaling in breast cancer, the molecular mechanisms involved, and its biological and therapeutic impact.

PP2A plays a key role in inflammation and cancer through tristetraprolin activation

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 …

Deregulation of miR-92a in locally advanced rectal cancer.

We read with interest the recent article by Pelossof et al. (2016), reporting a comparison between locally advanced rectal cancer (LARC) samples and normal rectal mucosa using an integrative genomic analysis that included microRNA and mRNA expression profiles and copy number alterations. Of importance, miR-92a was upregulated in LARC specimens and correlated inversely with the expression levels of its putative targets IQGAP2 and KLF4. These findings are consistent with previous work suggesting an oncogenic role of miR-92a in colorectal cancer (CRC) (Tsuchida et al., 2011; Yamada et al., 2013; Zhou et al., 2013). Despite the interesting data, the authors failed to perform a correct experimental validation and interpretation of their results. Western blot and luciferase assays, as also mentioned in their discussion, are required to demonstrate a direct regulation of IQGAP2 and KLF4 by miR-92a. Unfortunately, only changes in mRNA levels of these putative targets were analyzed after the ectopic modulation of miR92a expression. The authors claimed that a direct KLF4 regulation by miR-92a was not supported by their observations because they found no changes in KLF4 mRNA after miR-92a modulation. However, in the absence of a perfect match between microRNA and its seed region, the targeted mRNA is not degraded and changes in mRNA levels cannot be observed. Therefore, Western blot and luciferase assays have to be performed to evaluate KLF4 as a novel potential miR-92a target in forthcoming studies. Finally, it would be necessary to analyze the functional relevance of IQGAP2 and KLF4 deregulation compared to other previously described miR-92a targets such as BIM (Tsuchida et al., 2011), DKK-3 (Yamada et al., 2013), and PTEN (Zhang et al., 2014).

Analysis of Potential Alterations Affecting SETBP1 as a Novel Contributing Mechanism to Inhibit PP2A in Colorectal Cancer Patients

BackgroundThe 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.MethodsWe 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.ResultsWe 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.ConclusionsOur 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.