ERK5 mediates pro-tumorigenic phenotype in non-small lung cancer cells induced by PGE2.

Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) constituting approximately 84 % of all lung cancer cases. The role of inflammation in the initiation and progression of NSCLC tumors has been the focus of extensive research. Among the various inflammatory mediators, prostaglandin E2 (PGE2) plays a pivotal role in promoting the aggressiveness of epithelial tumors through multiple mechanisms, including the stimulation of growth, evasion of apoptosis, invasion, and induction of angiogenesis. The Extracellular signal-Regulated Kinase 5 (ERK5), the last discovered member among conventional mitogen-activated protein kinases (MAPK), is implicated in cancer-associated inflammation. In this study, we explored whether ERK5 is involved in the process of tumorigenesis induced by PGE2. Using A549 and PC9 NSCLC cell lines, we found that PGE2 triggers the activation of ERK5 via the EP1 receptor. Moreover, both genetic and pharmacological inhibition of ERK5 reduced PGE2-induced proliferation, migration, invasion and stemness of A549 and PC9 cells, indicating that ERK5 plays a critical role in PGE2-induced tumorigenesis. In summary, our study underscores the pivotal role of the PGE2/EP1/ERK5 axis in driving the malignancy of NSCLC cells in vitro. Targeting this axis holds promise as a potential avenue for developing novel therapeutic strategies aimed at controlling the advancement of NSCLC.

Importin subunit beta-1 mediates ERK5 nuclear translocation, and its inhibition synergizes with ERK5 kinase inhibitors in reducing cancer cell proliferation.

The mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 5 (ERK5) is emerging as a promising target in cancer. Indeed, alterations of the MEK5/ERK5 pathway are present in many types of cancer, including melanoma. One of the key events in MAPK signalling is MAPK nuclear translocation and its subsequent regulation of gene expression. Likewise, the effects of ERK5 in supporting cancer cell proliferation have been linked to its nuclear localization. Despite many processes regulating ERK5 nuclear translocation having been determined, the nuclear transporters involved have not yet been identified. Here, we investigated the role of importin subunit alpha (α importin) and importin subunit beta-1 (importin ß1) in ERK5 nuclear shuttling to identify additional targets for cancer treatment. Either importin ß1 knockdown or the α/ß1 importin inhibitor ivermectin reduced the nuclear amount of overexpressed and endogenous ERK5 in HEK293T and A375 melanoma cells, respectively. These results were confirmed in single-molecule microscopy in HeLa cells. Moreover, immunofluorescence analysis showed that ivermectin impairs epidermal growth factor (EGF)-induced ERK5 nuclear shuttling in HeLa cells. Both co-immunoprecipitation experiments and proximity ligation assay provided evidence that ERK5 and importin ß1 interact and that this interaction is further induced by EGF administration and prevented by ivermectin treatment. The combination of ivermectin and the ERK5 inhibitor AX15836 synergistically reduced cell viability and colony formation ability in A375 and HeLa cells and was more effective than single treatments in preventing the growth of A375 and HeLa spheroids. The increased reduction of cell viability upon the same combination was also observed in patient-derived metastatic melanoma cells. The combination of ivermectin and ERK5 inhibitors other than AX15836 provided similar effects on cell viability. The identification of importin ß1 as the nuclear transporter of ERK5 may be exploited for additional ERK5-inhibiting strategies for cancer therapy.

Latent-Transforming Growth Factor ß-Binding Protein 1/Transforming Growth Factor ß1 Complex Drives Antitumoral Effects upon ERK5 Targeting in Melanoma.

Melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. While available treatments have improved survival, long-term benefits are still unsatisfactory. The mitogen-activated protein kinase extracellular signal-regulated kinase 5 (ERK5) promotes melanoma growth, and ERK5 inhibition determines cellular senescence and the senescence-associated secretory phenotype. Here, latent-transforming growth factor ß-binding protein 1 (LTBP1) mRNA was found to be up-regulated in A375 and SK-Mel-5 BRAF V600E melanoma cells after ERK5 inhibition. In keeping with a key role of LTBP1 in regulating transforming growth factor ß (TGF-ß), TGF-ß1 protein levels were increased in lysates and conditioned media of ERK5-knockdown (KD) cells, and were reduced upon LTBP1 KD. Both LTBP1 and TGF-ß1 proteins were increased in melanoma xenografts in mice treated with the ERK5 inhibitor XMD8-92. Moreover, treatment with conditioned media from ERK5-KD melanoma cells reduced cell proliferation and invasiveness, and TGF-ß1-neutralizing antibodies impaired these effects. In silico data sets revealed that higher expression levels of both LTBP1 and TGF-ß1 mRNA were associated with better overall survival of melanoma patients. Increased LTBP1 or TGF-ß1 expression played a beneficial role in patients treated with anti-PD1 immunotherapy, making a possible immunosuppressive role of LTBP1/TGF-ß1 unlikely upon ERK5 inhibition. This study, therefore, identifies additional desirable effects of ERK5 targeting, providing evidence of an ERK5-dependent tumor-suppressive role of TGF-ß in melanoma.

Sphingosine 1-phosphate elicits a ROS-mediated proinflammatory response in human endometrial stromal cells via ERK5 activation.

Endometriosis is a chronic gynecological disease affecting ~10% women in the reproductive age characterized by the growth of endometrial glands and stroma outside the uterine cavity. The inflammatory process has a key role in the initiation and progression of the disorder. Currently, there are no available early diagnostic tests and therapy relies exclusively on symptomatic drugs, so that elucidation of the complex molecular mechanisms involved in the pathogenesis of endometriosis is an unmet need. The signaling of the bioactive sphingolipid sphingosine 1-phosphate (S1P) is deeply dysregulated in endometriosis. S1P modulates a variety of fundamental cellular processes, including inflammation, neo-angiogenesis, and immune responses acting mainly as ligand of a family of G-protein-coupled receptors named S1P receptors (S1PR), S1P1-5 . Here, we demonstrated that the mitogen-activated protein kinase ERK5, that is expressed in endometriotic lesions as determined by quantitative PCR, is activated by S1P in human endometrial stromal cells. S1P-induced ERK5 activation was shown to be triggered by S1P1/3 receptors via a SFK/MEK5-dependent axis. S1P-induced ERK5 activation was, in turn, responsible for the increase of reactive oxygen species and proinflammatory cytokine expression in human endometrial stromal cells. The present findings indicate that the S1P signaling, via ERK5 activation, supports a proinflammatory response in the endometrium and establish the rationale for the exploitation of innovative therapeutic targets for endometriosis.

Pathophysiological Impact of the MEK5/ERK5 Pathway in Oxidative Stress.

Oxidative stress regulates many physiological and pathological processes. Indeed, a low increase in the basal level of reactive oxygen species (ROS) is essential for various cellular functions, including signal transduction, gene expression, cell survival or death, as well as antioxidant capacity. However, if the amount of generated ROS overcomes the antioxidant capacity, excessive ROS results in cellular dysfunctions as a consequence of damage to cellular components, including DNA, lipids and proteins, and may eventually lead to cell death or carcinogenesis. Both in vitro and in vivo investigations have shown that activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway is frequently involved in oxidative stress-elicited effects. In particular, accumulating evidence identified a prominent role of this pathway in the anti-oxidative response. In this respect, activation of krüppel-like factor 2/4 and nuclear factor erythroid 2-related factor 2 emerged among the most frequent events in ERK5-mediated response to oxidative stress. This review summarizes what is known about the role of the MEK5/ERK5 pathway in the response to oxidative stress in pathophysiological contexts within the cardiovascular, respiratory, lymphohematopoietic, urinary and central nervous systems. The possible beneficial or detrimental effects exerted by the MEK5/ERK5 pathway in the above systems are also discussed.

MAPK15 protects from oxidative stress-dependent cellular senescence by inducing the mitophagic process.

Mitochondria are the major source of reactive oxygen species (ROS), whose aberrant production by dysfunctional mitochondria leads to oxidative stress, thus contributing to aging as well as neurodegenerative disorders and cancer. Cells efficiently eliminate damaged mitochondria through a selective type of autophagy, named mitophagy. Here, we demonstrate the involvement of the atypical MAP kinase family member MAPK15 in cellular senescence, by preserving mitochondrial quality, thanks to its ability to control mitophagy and, therefore, prevent oxidative stress. We indeed demonstrate that reduced MAPK15 expression strongly decreases mitochondrial respiration and ATP production, while increasing mitochondrial ROS levels. We show that MAPK15 controls the mitophagic process by stimulating ULK1-dependent PRKN Ser108 phosphorylation and inducing the recruitment of damaged mitochondria to autophagosomal and lysosomal compartments, thus leading to a reduction of their mass, but also by participating in the reorganization of the mitochondrial network that usually anticipates their disposal. Consequently, MAPK15-dependent mitophagy protects cells from accumulating nuclear DNA damage due to mitochondrial ROS and, consequently, from senescence deriving from this chronic DNA insult. Indeed, we ultimately demonstrate that MAPK15 protects primary human airway epithelial cells from senescence, establishing a new specific role for MAPK15 in controlling mitochondrial fitness by efficient disposal of old and damaged organelles and suggesting this kinase as a new potential therapeutic target in diverse age-associated human diseases.

Targeting MAPK in Cancer 2.0.

Mitogen-activated protein kinase (MAPK) pathways are prominently involved in the onset and progression of cancer [...].

Macrophage MerTK promotes profibrogenic cross-talk with hepatic stellate cells via soluble mediators.

BACKGROUND & AIMS: Activation of Kupffer cells and recruitment of monocytes are key events in fibrogenesis. These cells release soluble mediators which induce the activation of hepatic stellate cells (HSCs), the main fibrogenic cell type within the liver. Mer tyrosine kinase (MerTK) signaling regulates multiple processes in macrophages and has been implicated in the pathogenesis of non-alcoholic steatohepatitis-related fibrosis. In this study, we explored if MerTK activation in macrophages influences the profibrogenic phenotype of HSCs. METHODS: Macrophages were derived from THP-1 cells or differentiated from peripheral blood monocytes towards MerTK+/CD206+/CD163+/CD209- macrophages. The role of MerTK was assessed by pharmacologic and genetic inhibition. HSC migration was determined in Boyden chambers, viability was measured by the MTT assay, and proliferation was evaluated by the BrdU incorporation assay. RESULTS: Gas-6 induced MerTK phosphorylation and Akt activation in macrophages, and these effects were inhibited by UNC569. During polarization, MerTK+/CD206+/CD163+/CD209- macrophages exhibited activation of STAT3, ERK1/2, p38 and increased expression of VEGF-A. Activation of MerTK in THP-1 macrophages induced a secretome which promoted a significant increase in migration, proliferation, viability and expression of profibrogenic factors in HSCs. Similarly, conditioned medium from MerTK+ macrophages induced a significant increase in cell migration, proliferation, STAT3 and p38 phosphorylation and upregulation of IL-8 expression in HSCs. Moreover, conditioned medium from Gas-6-stimulated Kupffer cells induced a significant increase in HSC proliferation. These effects were specifically related to MerTK expression and activity in macrophages, as indicated by pharmacologic inhibition and knockdown experiments. CONCLUSIONS: MerTK activation in macrophages modifies the secretome to promote profibrogenic features in HSCs, implicating this receptor in the pathogenesis of hepatic fibrosis. LAY SUMMARY: Fibrosis represents the process of scarring occurring in patients with chronic liver diseases. This process depends on production of scar tissue components by a specific cell type, named hepatic stellate cells, and is regulated by interaction with other cells. Herein, we show that activation of MerTK, a receptor present in a population of macrophages, causes the production of factors that act on hepatic stellate cells, increasing their ability to produce scar tissue.

Lactate Maintains BCR/Abl Expression and Signaling in Chronic Myeloid Leukemia Cells Under Nutrient Restriction.

This study was directed to deepen the effects of nutrient shortage on BCR/Ablprotein expression and signaling in chronic myeloid leukemia (CML) cells. The backbone of the study was cell culture in medium lacking glucose, the consumption of which we had previously shown to drive BCR/Ablprotein suppression, and glutamine, the other main nutrient besides glucose. In this context, we focused on the role of lactate, the main by-product of glucose metabolism under conditions of rapid cell growth, in particular as a modulator of the maintenance of CML stem/progenitor cell potential, a crucial determinant of disease course and relapse of disease. The results obtained indicated that lactate is a powerful surrogate of glucose to prevent the suppression of BCR/Abl signaling and is therefore capable to maintain BCR/Abl-dependent CML stem/progenitor cell potential. A number of metabolism-related functional and phenotypical features of CML cells were also determined. Among these, we focused on the effect of lactate on oxygen consumption rate, the dependence of this effect on the cell surface lactate carrier MCT-1, and the relationship of the lactate effect to pyruvate and to the activity of mitochondrial pyruvate carrier.

Inhibition of ERK5 Elicits Cellular Senescence in Melanoma via the Cyclin-Dependent Kinase Inhibitor p21.

Melanoma is the deadliest skin cancer with a very poor prognosis in advanced stages. Although targeted and immune therapies have improved survival, not all patients benefit from these treatments. The mitogen-activated protein kinase ERK5 supports the growth of melanoma cells in vitro and in vivo. However, ERK5 inhibition results in cell-cycle arrest rather than appreciable apoptosis. To clarify the role of ERK5 in melanoma growth, we performed transcriptomic analyses following ERK5 knockdown in melanoma cells expressing BRAFV600E and found that cellular senescence was among the most affected processes. In melanoma cells expressing either wild-type or mutant (V600E) BRAF, both genetic and pharmacologic inhibition of ERK5 elicited cellular senescence, as observed by a marked increase in senescence-associated ß-galactosidase activity and p21 expression. In addition, depletion of ERK5 from melanoma cells resulted in increased levels of CXCL1, CXCL8, and CCL20, proteins typically involved in the senescence-associated secretory phenotype. Knockdown of p21 suppressed the induction of cellular senescence by ERK5 blockade, pointing to p21 as a key mediator of this process. In vivo, ERK5 knockdown or inhibition with XMD8-92 in melanoma xenografts promoted cellular senescence. Based on these results, small-molecule compounds targeting ERK5 constitute a rational series of prosenescence drugs that may be exploited for melanoma treatment. SIGNIFICANCE: This study shows that targeting ERK5 induces p21-mediated cellular senescence in melanoma, identifying a prosenescence effect of ERK5 inhibitors that may be exploited for melanoma treatment.

Hepatocyte-Specific Deletion of HIF2α Prevents NASH-Related Liver Carcinogenesis by Decreasing Cancer Cell Proliferation.

BACKGROUND & AIMS: Hypoxia and hypoxia-inducible factors (HIFs) are involved in chronic liver disease progression. We previously showed that hepatocyte HIF-2α activation contributed significantly to nonalcoholic fatty liver disease progression in experimental animals and human patients. In this study, using an appropriate genetic murine model, we mechanistically investigated the involvement of hepatocyte HIF-2α in experimental nonalcoholic steatohepatitis (NASH)-related carcinogenesis. METHODS: The role of HIF-2α was investigated by morphologic, cellular, and molecular biology approaches in the following: (1) mice carrying hepatocyte-specific deletion of HIF-2α (HIF-2α-/- mice) undergoing a NASH-related protocol of hepatocarcinogenesis; (2) HepG2 cells stably transfected to overexpress HIF-2α; and (3) liver specimens from NASH patients with hepatocellular carcinoma. RESULTS: Mice carrying hepatocyte-specific deletion of HIF-2α (hHIF-2α-/-) showed a significant decrease in the volume and number of liver tumors compared with wild-type littermates. These effects did not involve HIF-1α changes and were associated with a decrease of cell proliferation markers proliferating cell nuclear antigen and Ki67. In both human and rodent nonalcoholic fatty liver disease-related tumors, HIF-2α levels were strictly associated with hepatocyte production of SerpinB3, a mediator previously shown to stimulate liver cancer cell proliferation through the Hippo/Yes-associated protein (YAP)/c-Myc pathway. Consistently, we observed positive correlations between the transcripts of HIF-2α, YAP, and c-Myc in individual hepatocellular carcinoma tumor masses, while HIF-2α deletion down-modulated c-Myc and YAP expression without affecting extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and AKT-dependent signaling. In vitro data confirmed that HIF-2α overexpression induced HepG2 cell proliferation through YAP-mediated mechanisms. CONCLUSIONS: These results indicate that the activation of HIF-2α in hepatocytes has a critical role in liver carcinogenesis during NASH progression, suggesting that HIF-2α-blocking agents may serve as novel putative therapeutic tools.

The Hedgehog-GLI Pathway Regulates MEK5-ERK5 Expression and Activation in Melanoma Cells.

Malignant melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. We recently showed that the extracellular signal-regulated kinase 5 (ERK5), encoded by the MAPK7 gene, plays a pivotal role in melanoma by regulating cell functions necessary for tumour development, such as proliferation. Hedgehog-GLI signalling is constitutively active in melanoma and is required for proliferation. However, no data are available in literature about a possible interplay between Hedgehog-GLI and ERK5 pathways. Here, we show that hyperactivation of the Hedgehog-GLI pathway by genetic inhibition of the negative regulator Patched 1 increases the amount of ERK5 mRNA and protein. Chromatin immunoprecipitation showed that GLI1, the major downstream effector of Hedgehog-GLI signalling, binds to a functional non-canonical GLI consensus sequence at the MAPK7 promoter. Furthermore, we found that ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation, and that the combination of GLI and ERK5 inhibitors is more effective than single treatments in reducing cell viability and colony formation ability in melanoma cells. Together, these findings led to the identification of a novel Hedgehog-GLI-ERK5 axis that regulates melanoma cell growth, and shed light on new functions of ERK5, paving the way for new therapeutic options in melanoma and other neoplasms with active Hedgehog-GLI and ERK5 pathways.

Glutamine Availability Controls BCR/Abl Protein Expression and Functional Phenotype of Chronic Myeloid Leukemia Cells Endowed with Stem/Progenitor Cell Potential.

This study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective.

Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA. APPROACH AND RESULTS: ERK5 expression was detected in two established (HuCCT-1 and CCLP-1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT-1 and CCLP-1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5-silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP-1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA. CONCLUSIONS: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment.

Playing the Whack-A-Mole Game: ERK5 Activation Emerges Among the Resistance Mechanisms to RAF-MEK1/2-ERK1/2- Targeted Therapy.

Molecularly tailored therapies have opened a new era, chronic myeloid leukemia being the ideal example, in the treatment of cancer. However, available therapeutic options are still unsatisfactory in many types of cancer, and often fail due to the occurrence of resistance mechanisms. With regard to small-molecule compounds targeting the components of the Mitogen-Activated Protein Kinase (MAPK) cascade RAF-MEK1/2-ERK1/2, these drugs may result ineffective as a consequence of the activation of compensatory pro-survival/proliferative signals, including receptor tyrosine kinases, PI3K, as well as other components of the MAPK family such as TPL2/COT. The MAPK ERK5 has been identified as a key signaling molecule in the biology of several types of cancer. In this review, we report pieces of evidence regarding the activation of the MEK5-ERK5 pathway as a resistance mechanism to RAF-MEK1/2-ERK1/2 inhibitors. We also highlight the known and possible mechanisms underlying the cross-talks between the ERK1/2 and the ERK5 pathways, the characterization of which is of great importance to maximize, in the future, the impact of RAF-MEK1/2-ERK1/2 targeting. Finally, we emphasize the need of developing additional therapeutically relevant MEK5-ERK5 inhibitors to be used for combined treatments, thus preventing the onset of resistance to cancer therapies relying on RAF-MEK1/2-ERK1/2 inhibitors.

Enhanced Antitumoral Activity and Photoacoustic Imaging Properties of AuNP-Enriched Endothelial Colony Forming Cells on Melanoma.

Near infrared (NIR)-resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP-loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC-loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.

In Vitro Comparison of the Effects of Imatinib and Ponatinib on Chronic Myeloid Leukemia Progenitor/Stem Cell Features.

BACKGROUND: The development of molecularly tailored therapeutic agents such as the BCR/ABL-active tyrosine kinase inhibitors (TKi) resulted in an excellent treatment option for chronic myeloid leukemia (CML) patients. However, following TKi discontinuation, disease relapses in 40-60% of patients, an occurrence very likely due to the persistence of leukemic stem cells that are scarcely sensitive to TKi. Nevertheless, TKi are still the only current treatment option for CML patients. OBJECTIVE: The aim of this study was to compare the effects of TKi belonging to different generations, imatinib and ponatinib (first and third generation, respectively), on progenitor/stem cell expansion potential and markers. PATIENTS AND METHODS: We used stabilized CML cell lines (KCL22, K562 and LAMA-84 cells), taking advantage of the previous demonstration of ours that cell lines contain cell subsets endowed with progenitor/stem cell properties. Primary cells explanted from CML patients were also used. The effects of TKi on the expression of stem cell related genes were compared by quantitative PCR. Flow cytometry was performed to evaluate aldehyde-dehydrogenase (ALDH) activity and the expression of cluster of differentiation (CD) cell surface hematopoietic stem cell markers. Progenitor/stem cell potential was estimated by serial colony formation ability (CFA) assay. RESULTS: Ponatinib was more effective than imatinib for the reduction of cells with ALDH activity and progenitor/stem cell potential of CML patient-derived cells and cell lines. Furthermore, ponatinib was more effective than imatinib in reducing the percentage of CD26-expressing cells in primary CML cells, whereas imatinib and ponatinib showed similar efficacy on KCL22 cells. Both drugs strongly upregulated NANOG and SOX2 in CML cell lines, but in KCL22 cells this upregulation was significantly lower with ponatinib than with imatinib, an outcome compatible with a lower level of enrichment of the stem cell compartment upon ponatinib treatment. CONCLUSION: Ponatinib seems to target CML progenitor/stem cells better than imatinib.

Beyond Kinase Activity: ERK5 Nucleo-Cytoplasmic Shuttling as a Novel Target for Anticancer Therapy.

The importance of mitogen-activated protein kinases (MAPK) in human pathology is underlined by the relevance of abnormalities of MAPK-related signaling pathways to a number of different diseases, including inflammatory disorders and cancer. One of the key events in MAPK signaling, especially with respect to pro-proliferative effects that are crucial for the onset and progression of cancer, is MAPK nuclear translocation and its role in the regulation of gene expression. The extracellular signal-regulated kinase 5 (ERK5) is the most recently discovered classical MAPK and it is emerging as a possible target for cancer treatment. The bigger size of ERK5 when compared to other MAPK enables multiple levels of regulation of its expression and activity. In particular, the phosphorylation of kinase domain and C-terminus, as well as post-translational modifications and chaperone binding, are involved in ERK5 regulation. Likewise, different mechanisms control ERK5 nucleo-cytoplasmic shuttling, underscoring the key role of ERK5 in the nuclear compartment. In this review, we will focus on the mechanisms involved in ERK5 trafficking between cytoplasm and nucleus, and discuss how these processes might be exploited to design new strategies for cancer treatment.

The protein kinase CK2 contributes to the malignant phenotype of cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a particularly aggressive hepatobiliary malignancy, for which the molecular mechanisms underlying the malignant phenotype are still poorly understood, and novel and effective therapeutic strategies are limited. The pro-survival protein kinase CK2 is frequently overexpressed in cancer and is receiving increasing interest as an anti-tumor drug target. Its precise role in CCA biology is still largely unknown. Here we show that expression of the CK2α and α' catalytic subunits and of the ß regulatory subunit is increased in human CCA samples. Increased expression of CK2 subunits was shown in CCA cell lines compared to non-transformed cholangiocytes. We used chemical inhibition of CK2 and genetic modification by CRISPR/Cas9 to explore the contribution of CK2 to the malignant phenotype of CCA cells. Disruption of CK2 activity results in cell death through apoptosis, reduced invasion and migration potential, and G0/G1 cell cycle arrest. Importantly, CCA cells with a reduced CK2 activity are more sensitive to chemotherapy. Altogether, our results demonstrate that CK2 significantly contributes to increased proliferative potential and augmented growth of CCA cells and indicate the rationale for its targeting as a promising pharmacologic strategy for cholangiocarcinoma.