Acidic pH is an important feature of tumor microenvironment and a major determinant of tumor progression. We reported that cancer cells upregulate autophagy as a survival mechanism to acidic stress. Inhibition of autophagy by administration of chloroquine (CQ) in combination anticancer therapies is currently evaluated in clinical trials. We observed in 3 different human cancer cell lines cultured at acidic pH that autophagic flux is not blocked by CQ. This was consistent with a complete resistance to CQ toxicity in cells cultured in acidic conditions. Conversely, the autophagy-inhibiting activity of Lys-01, a novel CQ derivative, was still detectable at low pH. The lack of CQ activity was likely dependent on a dramatically reduced cellular uptake at acidic pH. Using cell lines stably adapted to chronic acidosis we could confirm that CQ lack of activity was merely caused by acidic pH. Moreover, unlike CQ, Lys-01 was able to kill low pH-adapted cell lines, although higher concentrations were required as compared with cells cultured at normal pH conditions. Notably, buffering medium pH in low pH-adapted cell lines reverted CQ resistance. In vivo analysis of tumors treated with CQ showed that accumulation of strong LC3 signals was observed only in normoxic areas but not in hypoxic/acidic regions. Our observations suggest that targeting autophagy in the tumor environment by CQ may be limited to well-perfused regions but not achieved in acidic regions, predicting possible limitations in efficacy of CQ in antitumor therapies.
Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.
A reciprocal translocation of the ABL1 gene to the BCR gene results in the expression of the oncogenic BCR-ABL1 fusion protein, which characterizes human chronic myeloid leukemia (CML), a myeloproliferative disorder considered invariably fatal until the introduction of the imatinib family of tyrosine kinase inhibitors (TKI). Nonetheless, insensitivity of CML stem cells to TKI treatment and intrinsic or acquired resistance are still frequent causes for disease persistence and blastic phase progression experienced in patients after initial successful therapies. Here, we investigated a possible role for the MAPK15/ERK8 kinase in BCR-ABL1-dependent autophagy, a key process for oncogene-induced leukemogenesis. In this context, we showed the ability of MAPK15 to physically recruit the oncogene to autophagic vesicles, confirming our hypothesis of a biologically relevant role for this MAP kinase in signal transduction by this oncogene. Indeed, by modeling BCR-ABL1 signaling in HeLa cells and taking advantage of a physiologically relevant model for human CML, i.e. K562 cells, we demonstrated that BCR-ABL1-induced autophagy is mediated by MAPK15 through its ability to interact with LC3-family proteins, in a LIR-dependent manner. Interestingly, we were also able to interfere with BCR-ABL1-induced autophagy by a pharmacological approach aimed at inhibiting MAPK15, opening the possibility of acting on this kinase to affect autophagy and diseases depending on this cellular function. Indeed, to support the feasibility of this approach, we demonstrated that depletion of endogenous MAPK15 expression inhibited BCR-ABL1-dependent cell proliferation, in vitro, and tumor formation, in vivo, therefore providing a novel “druggable” link between BCR-ABL1 and human CML.
ERK8 (MAPK15) is a large MAP kinase already implicated in the regulation of the functions of different nuclear receptors and in cellular proliferation and transformation. Here, we identify ERR␣ as a novel ERK8-interacting protein. As a consequence of such interaction, ERK8 induces CRM1-dependent translocation of ERR␣ to the cytoplasm and inhibits its transcriptional activity. Also, we identify in ERK8 two LXXLL motifs, typical of agonistbound nuclear receptor corepressors, as necessary features for this MAP kinase to interact with ERR␣ and to regulate its cellular localization and transcriptional activity. Ultimately, we demonstrate that ERK8 is able to counteract, in immortalized human mammary cells, ERR␣ activation induced by the EGF receptor pathway, often deregulated in breast cancer. Altogether, these results reveal a novel function for ERK8 as a bona fide ERR␣ corepressor, involved in control of its cellular localization by nuclear exclusion, and suggest a key role for this MAP kinase in the regulation of the biological activities of this nuclear receptor.Mitogen-activated protein (MAP) kinases are a family of proline-directed serine/threonine kinases expressed in all eukaryotic cells, from yeast to human, and involved in key signaling pathways regulating cell proliferation, differentiation, apoptosis, and stress response (1, 2). ERK8 is the last identified member of the MAP kinase family. Along with a typical MAP kinase domain, it possesses a peculiarly long, unique C-terminal domain (3). Its activity can be modulated by serum, DNA damage, and activated human oncogenes such as BCR/ABL and RET/PTC3 (3-5). Still, although ERK8 has the classical signature Thr-Glu-Tyr activation motif of signal-dependent kinases such as ERK1, ERK2, and ERK5, it is not significantly activated by many extracellular stimuli that typically impinge on MAP kinases, and no MAP kinase kinase (MAPKK) has been identified that regulates its activity (6).2 Interestingly, ERK8 has been recently involved in the transformation of human colon cancer cells (7) and in the maintenance of genomic integrity, by inhibiting proliferating cell nuclear antigen (PCNA) degradation (8). Recent data have also shown the ability of ERK8 to reduce the activity of nuclear receptors such as androgen and glucocorticoid receptors (9, 10). Moreover, ERK8 strongly enhances ubiquitin-dependent degradation of estrogen receptor ␣ (ER␣), 3 and loss of its expression has been correlated to breast cancer progression and increased ER␣ levels in mammary tumors (9). However, its targets and downstream effectors still remain almost completely unknown. Likewise, its biological functions have yet to be unraveled. ERR␣ (NR3B1) was the first "orphan" nuclear receptor to be identified, on the basis of its similarity to ER␣ (11). It is a key regulator of mitochondrial biogenesis (12), energy metabolism (13), and bone formation and maintenance (14). ERR␣ binds to DNA and regulates transcription through specific ERR response elements (ERREs), also named SF-1 response elements (SFR...
Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest.To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase.In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a “stress support” autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors.
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