Targeted Avenues for Cancer Treatment: The MEK5-ERK5 Signaling Pathway.

Twenty years have passed since extracellular signal-regulated kinase 5 (ERK5) and its upstream activator, mitogen-activated protein kinase 5 (MEK5), first emerged onto the cancer research scene. Although we have come a long way in defining the liaison between dysregulated MEK5-ERK5 signaling and the pathogenesis of epithelial and nonepithelial malignancies, selective targeting of this unique pathway remains elusive. Here, we provide an updated review of the existing evidence for a correlation between aberrant MEK5-ERK5 (phospho)proteomic/transcriptomic profiles, aggressive cancer states, and poor patient outcomes. We then focus on emerging insights from preclinical models regarding the relevance of upregulated ERK5 activity in promoting tumor growth, metastasis, therapy resistance, undifferentiated traits, and immunosuppression, highlighting the opportunities, prospects, and challenges of selectively blocking this cascade for antineoplastic treatment and chemosensitization.

MEK5/ERK5 activation regulates colon cancer stem-like cell properties.

Colon cancer has been proposed to be sustained by a small subpopulation of stem-like cells with unique properties allowing them to survive conventional therapies and drive tumor recurrence. Identification of targetable signaling pathways contributing to malignant stem-like cell maintenance may therefore translate into new therapeutic strategies to overcome drug resistance. Here we demonstrated that MEK5/ERK5 signaling activation is associated with stem-like malignant phenotypes. Conversely, using a panel of cell line-derived three-dimensional models, we showed that ERK5 inhibition markedly suppresses the molecular and functional features of colon cancer stem-like cells. Particularly, pharmacological inhibition of ERK5 using XMD8-92 reduced the rate of primary and secondary sphere formation, the expression of pluripotency transcription factors SOX2, NANOG, and OCT4, and the proportion of tumor cells with increased ALDH activity. Notably, this was further associated with increased sensitivity to 5-fluorouracil-based chemotherapy. Mechanistically, ERK5 inhibition resulted in decreased IL-8 expression and NF-κB transcriptional activity, suggesting a possible ERK5/NF-κB/IL-8 signaling axis regulating stem-like cell malignancy. Taken together, our results provide proof of principle that ERK5-targeted inhibition may be a promising therapeutic approach to eliminate drug-resistant cancer stem-like cells and improve colon cancer treatment.

The histone deacetylase inhibitor panobinostat is a potent antitumor agent in canine diffuse large B-cell lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common causes of cancer-related death in the United States and Europe. Although the outcome of NHL patients has improved over the last years with current therapies, the rate of mortality is still high. A plethora of new drugs is entering clinical development for NHL treatment; however, the approval of new treatments remains low due in part to the paucity of clinically relevant models for validation. Canine lymphoma shares remarkable similarities with its human counterpart, making the dog an excellent animal model to explore novel therapeutic molecules and approaches. Histone deacetylase inhibitors (HDACis) have emerged as a powerful new class of anti-cancer drugs for human therapy. To investigate HDACi antitumor properties on canine diffuse large B-cell lymphoma, a panel of seven HDACi compounds (CI-994, panobinostat, SBHA, SAHA, scriptaid, trichostatin A and tubacin) was screened on CLBL-1 canine B-cell lymphoma cell line. Our results demonstrated that all HDACis tested exhibited dose-dependent inhibitory effects on proliferation of CLBL-1 cells, while promoting increased H3 histone acetylation. Amongst all HDACis studied, panobinostat proved to be the most promising compound and was selected for further in vitro and in vivo evaluation. Panobinostat cytotoxicity was linked to H3 histone and α-tubulin acetylation, and to apoptosis induction. Importantly, panobinostat efficiently inhibited CLBL-1 xenograft tumor growth, and strongly induced acetylation of H3 histone and apoptosis in vivo. In conclusion, these results provide new data validating HDACis and, especially, panobinostat as a novel anti-cancer therapy for veterinary applications, while contributing to comparative oncology.

Convergence of miR-143 overexpression, oxidative stress and cell death in HCT116 human colon cancer cells.

MicroRNAs (miRNAs) regulate a wide variety of biological processes, including tumourigenesis. Altered miRNA expression is associated with deregulation of signalling pathways, which in turn cause abnormal cell growth and de-differentiation, contributing to cancer. miR-143 and miR-145 are anti-tumourigenic and influence the sensitivity of tumour cells to chemotherapy and targeted therapy. Comparative proteomic analysis was performed in HCT116 human colon cancer cells stably transduced with miR-143 or miR-145. Immunoblotting analysis validated the proteomic data in stable and transient miRNA overexpression conditions in human colon cancer cells. We show that approximately 100 proteins are differentially expressed in HCT116 human colon cancer cells stably transduced with miR-143 or miR-145 compared to Empty control cells. Further, Gene Ontology and pathway enrichment analysis indicated that proteins involved in specific cell signalling pathways such as cell death, response to oxidative stress, and protein folding might be modulated by these miRNAs. In particular, antioxidant enzyme superoxide dismutase 1 (SOD1) was downregulated by stable expression of either miR-143 or miR-145. Further, SOD1 gain-of-function experiments rescued cells from miR-143-induced oxidative stress. Moreover, miR-143 overexpression increased oxaliplatin-induced apoptosis associated with reactive oxygen species generation, which was abrogated by genetic and pharmacological inhibition of oxidative stress. Overall, miR-143 might circumvent resistance of colon cancer cells to oxaliplatin via increased oxidative stress in HCT116 human colon cancer cells.

miRNAs as Modulators of EGFR Therapy in Colorectal Cancer.

Drug resistance is a serious impediment to the treatment of cancer. The use of anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies in patients with metastatic colorectal cancer is guided by the presence of activating point mutations in KRAS and NRAS genes in the primary tumour. However, RAS wild-type status is still not sufficient to guarantee response to cetuximab and panitumumab, with response rates limited to 70% for combinations with multidrug chemotherapy. Therefore, additional mechanisms contributing to resistance are currently under investigation, and include genetic alterations and epigenetic mechanisms of resistance. In this regard, deregulation of miRNA expression profiles holds potential to unveil resistance and fuel the development of miRNA-based strategies to overcome EGFR-directed therapy limitations. We discuss current understanding of miRNA impact as modulators of EGFR therapy in patients with metastatic colorectal cancer and the future challenge of miRNAs in circulation as powerful non-invasive tools to monitor anti-EGFR therapy response and predict resistance.

MEK5/ERK5 signaling inhibition increases colon cancer cell sensitivity to 5-fluorouracil through a p53-dependent mechanism.

The MEK5/ERK5 signaling pathway is emerging as an important contributor to colon cancer onset, progression and metastasis; however, its relevance to chemotherapy resistance remains unknown. Here, we evaluated the impact of the MEK5/ERK5 cascade in colon cancer cell sensitivity to 5-fluorouracil (5-FU). Increased ERK5 expression was correlated with poor overall survival in colon cancer patients. In colon cancer cells, 5-FU exposure impaired endogenous KRAS/MEK5/ERK5 expression and/or activation. In turn, MEK5 constitutive activation reduced 5-FU-induced cytotoxicity. Using genetic and pharmacological approaches, we showed that ERK5 inhibition increased caspase-3/7 activity and apoptosis following 5-FU exposure. Mechanistically, this was further associated with increased p53 transcriptional activation of p21 and PUMA. In addition, ERK5 inhibition increased the response of HCT116 p53+/+ cells to 5-FU, but failed to sensitize HCT116 p53-/- cells to the cytotoxic effects of this chemotherapeutic agent, suggesting a p53-dependent axis mediating 5-FU sensitization. Finally, ERK5 inhibition using XMD8-92 was shown to increase the antitumor effects of 5-FU in a murine subcutaneous xenograft model, enhancing apoptosis while markedly reducing tumor growth. Collectively, our results suggest that ERK5-targeted inhibition provides a promising therapeutic approach to overcome resistance to 5-FU-based chemotherapy and improve colon cancer treatment.

New [(η(5)-C5H5)Ru(N-N)(PPh3)][PF6] compounds: colon anticancer activity and GLUT-mediated cellular uptake of carbohydrate-appended complexes.

Eight ruthenium(ii) compounds of the general formula [(η(5)-C5H5)Ru(N-N)(PPh3)][PF6] were rationally designed, exhibiting high cytotoxicity against HCT116 human colon cancer cells, with IC50 between 14.56 and 1.56 µM; importantly, compounds 5Ru and 6Ru are the first reported ruthenium glycoconjugates exploiting glucose transporters, widely overexpressed in cancer, for cellular uptake.

miR-143 or miR-145 overexpression increases cetuximab-mediated antibody-dependent cellular cytotoxicity in human colon cancer cells.

miR-143 and miR-145 are downregulated in colon cancer. Here, we tested the effect of restoring these miRNAs on sensitization to cetuximab in mutant KRAS (HCT116 and SW480) and wild-type KRAS (SW48) colon cancer cells. We evaluated cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and the modulation of signaling pathways involved in immune effector cell-mediated elimination of cancer cells. Stable miR-143 or miR-145 overexpression increased cell sensitivity to cetuximab, resulting in a significant increase of cetuximab-mediated ADCC independently of KRAS status. Importantly, HCT116 cells overexpressing these miRNAs triggered apoptosis in result of cetuximab-mediated ADCC, effected by peripheral blood mononuclear cells (p < 0.01). This was associated with increased apoptosis and caspase-3/7 activity, and reduced Bcl-2 protein expression (p < 0.01). In addition, caspase inhibition abrogated cetuximab-mediated ADCC in HCT116 cells overexpressing either miR-143 or miR-145 (p < 0.01). Furthermore, Bcl-2 silencing led to high level of cetuximab-mediated ADCC, compared to control siRNA (p < 0.05). Importantly, granzyme B inhibition, abrogated cetuximab-mediated ADCC, reducing caspase-3/7 activity (p < 0.01). Collectively, our data suggests that re-introduction of miR-143 or miR-145 may provide a new approach for development of therapeutic strategies to re-sensitize colon cancer cells to cetuximab by stimulating cetuximab-dependent ADCC to induce cell death.

Cyclopentadienyl-ruthenium(II) and iron(II) organometallic compounds with carbohydrate derivative ligands as good colorectal anticancer agents.

New ruthenium(II) and iron(II) organometallic compounds of general formula [(η(5)-C5H5)M(PP)Lc][PF6], bearing carbohydrate derivative ligands (Lc), were prepared and fully characterized and the crystal structures of five of those compounds were determined by X-ray diffraction studies. Cell viability of colon cancer HCT116 cell line was determined for a total of 23 organometallic compounds and SAR's data analysis within this library showed an interesting dependency of the cytotoxic activity on the carbohydrate moiety, linker, phosphane coligands, and metal center. More importantly, two compounds, 14Ru and 18Ru, matched oxaliplatin IC50 (0.45 µM), the standard metallodrug used in CC chemotherapeutics, and our leading compound 14Ru was shown to be significantly more cytotoxic than oxaliplatin to HCT116 cells, triggering higher levels of caspase-3 and -7 activity and apoptosis in a dose-dependent manner.

c-Jun N-terminal kinase 1/c-Jun activation of the p53/microRNA 34a/sirtuin 1 pathway contributes to apoptosis induced by deoxycholic acid in rat liver.

MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ursodeoxycholic acid, a hydrophilic bile acid, counteracts the miR-34a/sirtuin 1 (SIRT1)/p53 pathway, activated in the liver of nonalcoholic steatohepatitis (NASH) patients. In contrast, hydrophobic bile acids, particularly deoxycholic acid (DCA), activate apoptosis and are increased in NASH. We evaluated whether DCA-induced apoptosis of rat hepatocytes occurs via miR-34a-dependent pathways and whether they connect with c-Jun N-terminal kinase (JNK) induction. DCA enhanced miR-34a/SIRT1/p53 proapoptotic signaling in a dose- and time-dependent manner. In turn, miR-34a inhibition and SIRT1 overexpression significantly rescued targeting of the miR-34a pathway and apoptosis by DCA. In addition, p53 overexpression activated the miR-34a/SIRT1/p53 pathway, further induced by DCA. DCA increased p53 expression as well as p53 transcriptional activation of PUMA and miR-34a itself, providing a functional mechanism for miR-34a activation. JNK1 and c-Jun were shown to be major targets of DCA, upstream of p53, in engaging the miR-34a pathway and apoptosis. Finally, activation of this JNK1/miR-34a proapoptotic circuit was also shown to occur in vivo in the rat liver. These results suggest that the JNK1/p53/miR-34a/SIRT1 pathway may represent an attractive pharmacological target for the development of new drugs to arrest metabolism- and apoptosis-related liver pathologies.

Efficient recovery of proteins from multiple source samples after TRIzol(®) or TRIzol(®)LS RNA extraction and long-term storage.

BACKGROUND: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol(®) and TRIzol(®)LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation. RESULTS: TRIzol(®)LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol(®) was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer's disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol(®)-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at -80°C until used for protein isolation.Simple modifications to the TRIzol(®) manufacturer's protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol(®) manufacturer's protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol(®)-chloroform fractions stored for up to 2 years at -80°C. CONCLUSIONS: We provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol(®) and TRIzol(®)LS compared to the manufacturer`s protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer's disease mice model, stored in TRIzol(®)-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol(®) manufacturer`s protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

Delivering the promise of miRNA cancer therapeutics.

MicroRNAs (miRNAs) are pivotal post-transcriptional gene expression regulators. These endogenous small non-coding RNAs aberrantly expressed in cancer have significant roles in tumorigenesis and progression. Currently, miRNAs are being pursued as diagnostic and prognostic biomarkers, and as therapeutic tools in cancer. miRNA modulation provides the unique ability to fine-tune multiple genes simultaneously, thereby regulating relevant signaling pathways involved in cell differentiation, proliferation and survival. This unique miRNA feature shifts the traditional one drug one target paradigm to a novel one drug multiple targets paradigm. We herein review in vivo strategies of miRNA modulator (mimic and/or inhibitor) delivery in cancer models, a subject that remains the key challenge to the establishment of this novel class of RNA therapeutics.