The extracellular signal-related kinases (ERKs) act as pleiotropic molecules in tumors, where they activate pro-survival pathways leading to cell proliferation and migration, as well as modulate apoptosis, differentiation, and senescence. Given its central role as sensor of extracellular signals, ERK transduction system is widely exploited by cancer cells subjected to environmental stresses, such as chemotherapy and anti-tumor activity of the host immune system. Aggressive tumors have a tremendous ability to adapt and survive in stressing and unfavorable conditions. The simultaneous resistance to chemotherapy and immune system responses is common, and ERK signaling plays a key role in both types of resistance. In this review, we dissect the main ERK-dependent mechanisms and feedback circuitries that simultaneously determine chemoresistance and immune-resistance/immune-escape in cancer cells. We discuss the pros and cons of targeting ERK signaling to induce chemo-immune-sensitization in refractory tumors.

Background Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis is poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells. Methods A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, was used. Results Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently noncytotoxic doses of temozolomide, which chemosensitized glioblastoma cells to doxorubicin, vinblastine, and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to an investigation of whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. Temozolomide-treated cells had increased methylation of the cytosine–phosphate–guanine islands in the Wnt3a gene promoter, decreased expression of Wnt3a, disrupted glycogen synthase-3 kinase/β-catenin axis, reduced transcriptional activation of ABCB1, and a lower amount and activity of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary, glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and ABCB1 levels. Conclusions Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation, and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cell population by downregulating Wnt3a signaling.

Glioblastoma is the most common and invasive primary tumor of the central nervous system and normally has a negative prognosis. Biodistribution in healthy animal models is an important preliminary study aimed at investigating the efficacy of chemotherapy, as it is mainly addressed towards residual cells after surgery in a region with an intact blood–brain barrier. Nanoparticles have emerged as versatile vectors that can overcome the blood–brain barrier. In this experimental work, solid lipid nanoparticles, prepared using fatty acid coacervation, have been loaded with an active lipophilic ester of cytotoxic drug methotrexate, and functionalized with either transferrin or insulin, two proteins whose receptors are abundantly expressed on the blood–brain barrier. Functionalization has been achieved by grafting a maleimide moiety onto the nanoparticle’s surface and exploiting its reactivity towards thiolated proteins. The nanoparticles have been tested in vitro on a blood–brain barrier cellular model and in vivo for biodistribution in Wistar rats. Drug metabolites, in particular 7-hydroxymethotrexate, have also been investigated in the animal model. The data obtained indicate that the functionalization of the nanoparticles improved their ability to overcome the blood–brain barrier when a PEG spacer between the proteins and the nanoparticle’s surface was used. This is probably because this method provided improved ligand–receptor interactions and selectivity for the target tissue.

Systemic treatment of malignant pleural mesothelioma (MPM) is moderately active for the intrinsic pharmacological resistance of MPM cell and its ability to induce an immune suppressive environment. Here we showed that the expression of bromodomain (BRD) proteins BRD2, BRD4 and BRD9 was significantly higher in human primary MPM cells compared to normal mesothelial cells (HMC). Nanomolar concentrations of bromodomain inhibitors (BBIs) JQ1 or OTX015 impaired patient-derived MPM cell proliferation and induced cell-cycle arrest without affecting apoptosis. Importantly, BBIs primed MPM cells for immunogenic cell death, by increasing extracellular release of ATP and HMGB1, and by promoting membrane exposure of calreticulin and ERp57. Accordingly, BBIs activated dendritic cell (DC)-mediated phagocytosis and expansion of CD8⁺ T-lymphocyte clones endorsed with antitumor cytotoxic activity. BBIs reduced the expression of the immune checkpoint ligand PD-L1 in MPM cells; while both CD8⁺ and CD4⁺ T-lymphocytes co-cultured with JQ1-treated MPM cells decreased PD-1 expression, suggesting a disruption of the immune-suppressive PD-L1/PD-1 axis. Additionally, BBIs reduced the expansion of myeloid-derived suppressor cells (MDSC) induced by MPM cells. Finally, a preclinical model of MPM confirmed that the anti-tumor efficacy of JQ1 was largely due to its ability to restore an immune-active environment, by increasing intra-tumor DC and CD8⁺ T-lymphocytes, and decreasing MDSC. Thereby, we propose that, among novel drugs, BBIs should be investigated for MPM treatment for their combined activity on both tumor cells and surrounding immune-environment.

Triple negative breast cancer (TNBC) easily develops resistance to the first-line drug doxorubicin, because of the high levels of the drug efflux transporter P-glycoprotein (Pgp) and the activation of pro-survival pathways dependent on endoplasmic reticulum (ER). Interfering with these mechanisms may overcome the resistance to doxorubicin, a still unmet need in TNBC. We analyzed a panel of human and murine breast cancer cells for their resistance to doxorubicin, Pgp expression, lysosome and proteasome activity, nitrite production, ER-dependent cell death and immunogenic cell death parameters. We evaluated the efficacy of genetic (C/EBP-β LIP induction) and pharmacological strategies (lysosome and proteasome inhibitors), in restoring the ER-dependent and immunogenic-dependent cell death induced by doxorubicin, in vitro and in syngeneic mice bearing chemoresistant TNBC. The results were analyzed by one-way analysis of variance test. We found that TNBC cells characterized by high levels of Pgp and resistance to doxorubicin, had low induction of the ER-dependent pro-apoptotic factor C/EBP-β LIP upon doxorubicin treatment and high activities of lysosome and proteasome that constitutively destroyed LIP. The combination of chloroquine and bortezomib restored doxorubicin sensitivity by activating multiple and interconnected mechanisms. First, chloroquine and bortezomib prevented C/EBP-β LIP degradation and activated LIP-dependent CHOP/TRB3/caspase 3 axis in response to doxorubicin. Second, C/EBP-β LIP down-regulated Pgp and up-regulated calreticulin that triggered the dendritic cell (DC)-mediated phagocytosis of tumor cell, followed by the activation of anti-tumor CD8+T-lymphocytes upon doxorubicin treatment. Third, chloroquine and bortezomib increased the endogenous production of nitric oxide that further induced C/EBP-β LIP and inhibited Pgp activity, enhancing doxorubicin’s cytotoxicity. In orthotopic models of resistant TNBC, intratumor C/EBP-β LIP induction - achieved by a specific expression vector or by chloroquine and bortezomib - effectively reduced tumor growth and Pgp expression, increased intra-tumor apoptosis and anti-tumor immune-infiltrate, rescuing the efficacy of doxorubicin. We suggest that preventing C/EBP-β LIP degradation by lysosome and proteasome inhibitors triggers multiple virtuous circuitries that restore ER-dependent apoptosis, down-regulate Pgp and re-activate the DC/CD8+T-lymphocytes response against TNBC. Lysosome and proteasome inhibitors associated with doxorubicin may overcome the resistance to the drug in TNBC.

The role of carbonic anhydrase XII (CAXII) in the chemoresistance of glioblastoma is unexplored. We found CAXII and P-glycoprotein (Pgp) coexpressed in neurospheres derived from 3 of 3 patients with different genetic backgrounds and low response to temozolomide (time to recurrence: 6–9 months). CAXII was necessary for the Pgp efflux of temozolomide and second-line chemotherapeutic drugs, determining chemoresistance in neurospheres. Psammaplin C, a potent inhibitor of CAXII, resensitized primary neurospheres to temozolomide by reducing temozolomide efflux via Pgp. This effect was independent of other known temozolomide resistance factors present in the patients. The overall survival in orthotopic patient-derived xenografts of temozolomide-resistant neurospheres, codosed with Psammaplin C and temozolomide, was significantly increased over temozolomide-treated (P < 0.05) and untreated animals (P < 0.02), without detectable signs of systemic toxicity. We propose that a CAXII inhibitor in combination with temozolomide may provide a new and effective approach to reverse chemoresistance in glioblastoma stem cells. This novel mechanism of action, via the interaction of CAXII and Pgp, ultimately blocks the efflux function of Pgp to improve glioblastoma patient outcomes.