Targeting tumor-associated macrophages to increase the efficacy of immune checkpoint inhibitors: a glimpse into novel therapeutic approaches for metastatic melanoma

Immune checkpoint inhibitors (ICIs) represent a promising therapeutic intervention for a variety of advanced/metastatic solid tumors, including melanoma, but in a large number of cases, patients fail to establish a sustained anti-tumor immunity and to achieve a long-lasting clinical benefit. Cells of the tumor micro-environment such as tumor-associated M2 macrophages (M2-TAMs) have been reported to limit the efficacy of immunotherapy, promoting tumor immune evasion and progression. Thus, strategies targeting M2-TAMs have been suggested to synergize with immune checkpoint blockade. This review recapitulates the molecular mechanisms by which M2-TAMs promote cancer immune evasion, with focus on the potential cross-talk between pharmacological interventions targeting M2-TAMs and ICIs for melanoma treatment

Platelet-derived growth factor C and calpain-3 are modulators of human melanoma cell invasiveness.

The molecular mechanisms responsible for the elevated metastatic potential of malignant melanoma are still not fully understood. In order to shed light on the molecules involved in the acquisition by melanoma of a highly aggressive phenotype, we compared the gene expression profiles of two cell clones derived from the human cutaneous metastatic melanoma cell line M14: a highly invasive clone (M14C2/MK18) and a clone (M14C2/C4) with low ability to invade the extracellular matrix (ECM). The highly invasive phenotype of M14C2/MK18 cells was correlated with overexpression of neuropilin-1, activation of a vascular endothelial growth factor (VEGF)-A/VEGFR-2 autocrine loop and secretion of matrix metalloprotease-2. Moreover, in an in vivo murine model, M14C2/MK18 cells displayed a higher growth rate as compared with M14C2/C4 cells, even though in vitro both clones possessed comparable proliferative potential. Microarray analysis in M14C2/MK18 cells showed a strong upregulation of platelet-derived growth factor (PDGF)-C, a cytokine that contributes to angiogenesis, and downregulation of calpain-3, a calcium-dependent thiol-protease that regulates specific signalling cascade components. Inhibition of PDGF-C with a specific antibody resulted in a significant decrease in ECM invasion by M14C2/MK18 cells, confirming the involvement of PDGF-C in melanoma cell invasiveness. Moreover, the PDGF-C transcript was found to be upregulated in a high percentage of human melanoma cell lines (17/20), whereas only low PDGF-C levels were detected in a few melanocytic cultures (2/6). By contrast, inhibition of calpain-3 activity in M14C2/C4 control cells, using a specific chemical inhibitor, markedly increased ECM invasion, strongly suggesting that downregulation of calpain-3 plays a role in the acquisition of a highly invasive phenotype. The results indicate that PDGF-C upregulation and calpain-3 downregulation are involved in the aggressiveness of malignant melanoma and suggest that modulators of these proteins or their downstream effectors may synergise with VEGF‑A therapies in combating tumour-associated angiogenesis and melanoma spread

At the cutting edge against cancer: A perspective on immunoproteasome and immune checkpoints modulation as a potential therapeutic intervention

Simple Summary:& nbsp;Immunoproteasome plays a key role in the generation of antigenic peptides. Immune checkpoints therapy is a front-line treatment of advanced/metastatic tumors, and to improve its efficacy, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is mandatory. The scope of this review is to offer a picture of the role of immunoproteasome in antigen presentation to fuel the hypothesis of novel therapeutic interventions based on the modulation of this proteolytic complex and immune checkpoints.Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers

Influence of MLH1 on colon cancer sensitivity to poly(ADP-ribose) polymerase inhibitor combined with irinotecan

Poly(ADP-ribose) polymerase inhibitors (PARPi) are currently evaluated in clinical trials in combination with topoisomerase I (Top1) inhibitors against a variety of cancers, including colon carcinoma. Since the mismatch repair component MLH1 is defective in 10-15% of colorectal cancers we have investigated whether MLH1 affects response to the Top1 inhibitor irinotecan, alone or in combination with PARPi. To this end, the colon cancer cell lines HCT116, carrying MLH1 mutations on chromosome 3 and HCT116 in which the wildtype MLH1 gene was replaced via chromosomal transfer (HCT116+3) or by transfection of the corresponding MLH1 cDNA (HCT116 1-2) were used. HCT116 cells or HCT116+3 cells stably silenced for PARP-1 expression were also analysed. The results of in vitro and in vivo experiments indicated that MLH1, together with low levels of Top1, contributed to colon cancer resistance to irinotecan. In the MLH1-proficient cells SN-38, the active metabolite of irinotecan, induced lower levels of DNA damage than in MLH1-deficient cells, as shown by the weaker induction of γ-H2AX and p53 phosphorylation. The presence of MLH1 contributed to induce of prompt Chk1 phosphorylation, restoring G2/M cell cycle checkpoint and repair of DNA damage. On the contrary, in the absence of MLH1, HCT116 cells showed minor Chk1 phosphorylation and underwent apoptosis. Remarkably, inhibition of PARP function by PARPi or by PARP-1 gene silencing always increased the antitumor activity of irinotecan, even in the presence of low PARP-1 expression

Clinical experience with CTLA-4 blockade for cancer immunotherapy: From the monospecific monoclonal antibody ipilimumab to probodies and bispecific molecules targeting the tumor microenvironment

The immune checkpoint cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an inhibitory regulator of T-cell mediated responses that has been investigated as target of monoclonal antibodies (mAbs) for cancer immunotherapy. The anti-CTLA-4 mAb ipilimumab represents the first immune checkpoint inhibitor that significantly improved overall survival in patients with unresectable/metastatic melanoma. The subsequent approved indications (often in the first-line setting) for melanoma and other advanced/metastatic solid tumors always require ipilimumab combination with nivolumab, an anti-programmed cell death protein 1 (PD-1) mAb. However, the improved clinical efficacy of the mAb combination is associated with increased immune-related adverse events, which might require treatment discontinuation even in responding patients. This drawback is expected to be overcome by the recent development of anti-CTLA-4 probodies proteolitycally activated in the tumor microenvironment and bispecific molecules targeting both CTLA-4 and PD-1, whose co-expression is characteristic of tumor-infiltrating T cells. These molecules would preferentially stimulate immune responses against the tumor, reducing toxicity toward normal tissues

Multiple sclerosis treatment and melanoma development

Therapy of multiple sclerosis (MS) with disease-modifying agents such as natalizumab or fingolimod has been associated with the development of cutaneous melanoma. Here we briefly revise literature data and report of a case of a 48-year old woman who developed a melanoma and several atypical naevi after sub sequential treatment with natalizumab (1 year) and fingolimod (7 years). By immunohistochemistry we observed the presence of T cells and leukocyte infiltration as well as of vascular endothelial growth factor (VEGF)-A expression in the patient melanoma biopsy. Then, we analyzed proliferation, migration and VEGF-A expression in three melanoma cell lines and found out that both natalizumab and fingolimod inhibited tumor cell proliferation but promoted or blocked cell migration depending on the cell line examined. VEGF-A secretion was augmented in one melanoma cell line only after fingolimod treatment. In conclusion, our in vitro data do not support the hypothesis of a direct action of natalizumab or fingolimod on melanoma progression but acting on the tumor microenvironment these treatments could indirectly favor melanoma evolution

Pdia3 expression in glioblastoma modulates macrophage/microglia pro-tumor activation

The glioblastoma (GB) microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs) that are still poorly characterized. A potential role on the mechanisms regulating GAM activity might be played by the endoplasmic reticulum protein ERp57/PDIA3 (protein disulfide-isomerase A3), the modulation of which has been reported in a variety of cancers. Moreover, by using The Cancer Genome Atlas database, we found that overexpression of PDIA3 correlated with about 55% reduction of overall survival of glioma patients. Therefore, we analyzed the expression of ERp57/PDIA3 using specimens obtained after surgery from 18 GB patients. Immunohistochemical analysis of tumor samples revealed ERp57/PDIA3 expression in GB cells as well as in GAMs. The ERp57/PDIA3 levels were higher in GAMs than in the microglia present in the surrounding parenchyma. Therefore, we studied the role of PDIA3 modulation in microglia\u2013glioma interaction, based on the ability of conditioned media collected from human GB cells to induce the activation of microglial cells. The results indicated that reduced PDIA3 expression/activity in GB cells significantly limited the microglia pro-tumor polarization towards the M2 phenotype and the production of pro-inflammatory factors. Our data support a role of PDIA3 expression in GB-mediated protumor activation of microglia

Clinical relevance of the transcriptional signature regulated by CDC42 in colorectal cancer.

CDC42 is an oncogenic Rho GTPase overexpressed in colorectal cancer (CRC). Although CDC42 has been shown to regulate gene transcription, the specific molecular mechanisms regulating the oncogenic ability of CDC42 remain unknown. Here, we have characterized the transcriptional networks governed by CDC42 in the CRC SW620 cell line using gene expression analysis. Our results establish that several cancer-related signaling pathways, including cell migration and cell proliferation, are regulated by CDC42. This transcriptional signature was validated in two large cohorts of CRC patients and its clinical relevance was also studied. We demonstrate that three CDC42-regulated genes offered a better prognostic value when combined with CDC42 compared to CDC42 alone. In particular, the concordant overexpression of CDC42 and silencing of the putative tumor suppressor gene CACNA2D2 dramatically improved the prognostic value. The CACNA2D2/CDC42 prognostic classifier was further validated in a third CRC cohort as well as in vitro and in vivo CRC models. Altogether, we show that CDC42 has an active oncogenic role in CRC via the transcriptional regulation of multiple cancer-related pathways and that CDC42-mediated silencing of CACNA2D2 is clinically relevant. Our results further support the use of CDC42 specific inhibitors for the treatment of the most aggressive types of CRC

VEGF-A/VEGFR-1 signalling and chemotherapy-induced neuropathic pain: therapeutic potential of a novel anti-VEGFR-1 monoclonal antibody

Abstract Background Neuropathic pain is a clinically relevant adverse effect of several anticancer drugs that markedly impairs patients’ quality of life and frequently leads to dose reduction or therapy discontinuation. The poor knowledge about the mechanisms involved in neuropathy development and pain chronicization, and the lack of effective therapies, make treatment of chemotherapy-induced neuropathic pain an unmet medical need. In this context, the vascular endothelial growth factor A (VEGF-A) has emerged as a candidate neuropathy hallmark and its decrease has been related to pain relief. In the present study, we have investigated the role of VEGF-A and its receptors, VEGFR-1 and VEGFR-2, in pain signalling and in chemotherapy-induced neuropathy establishment as well as the therapeutic potential of receptor blockade in the management of pain. Methods Behavioural and electrophysiological analyses were performed in an in vivo murine model, by using selective receptor agonists, blocking monoclonal antibodies or siRNA-mediated silencing of VEGF-A and VEGFRs. Expression of VEGF-A and VEGFR-1 in astrocytes and neurons was detected by immunofluorescence staining and confocal microscopy analysis. Results In mice, the intrathecal infusion of VEGF-A (VEGF165 isoforms) induced a dose-dependent noxious hypersensitivity and this effect was mediated by VEGFR-1. Consistently, electrophysiological studies indicated that VEGF-A strongly stimulated the spinal nociceptive neurons activity through VEGFR-1. In the dorsal horn of the spinal cord of animals affected by oxaliplatin-induced neuropathy, VEGF-A expression was increased in astrocytes while VEGFR-1 was mainly detected in neurons, suggesting a VEGF-A/VEGFR-1-mediated astrocyte-neuron cross-talk in neuropathic pain pathophysiology. Accordingly, the selective knockdown of astrocytic VEGF-A by intraspinal injection of shRNAmir blocked the development of oxaliplatin-induced neuropathic hyperalgesia and allodynia. Interestingly, both intrathecal and systemic administration of the novel anti-VEGFR-1 monoclonal antibody D16F7, endowed with anti-angiogenic and antitumor properties, reverted oxaliplatin-induced neuropathic pain. Besides, D16F7 effectively relieved hypersensitivity induced by other neurotoxic chemotherapeutic agents, such as paclitaxel and vincristine. Conclusions These data strongly support the role of the VEGF-A/VEGFR-1 system in mediating chemotherapy-induced neuropathic pain at the central nervous system level. Thus, treatment with the anti-VEGFR-1 mAb D16F7, besides exerting antitumor activity, might result in the additional advantage of attenuating neuropathic pain when combined with neurotoxic anticancer agents

VEGF-A/VEGFR-1 signalling and chemotherapy-induced neuropathic pain: therapeutic potential of a novel anti-VEGFR-1 monoclonal antibody

Background Neuropathic pain is a clinically relevant adverse effect of several anticancer drugs that markedly impairs patients' quality of life and frequently leads to dose reduction or therapy discontinuation. The poor knowledge about the mechanisms involved in neuropathy development and pain chronicization, and the lack of effective therapies, make treatment of chemotherapy-induced neuropathic pain an unmet medical need. In this context, the vascular endothelial growth factor A (VEGF-A) has emerged as a candidate neuropathy hallmark and its decrease has been related to pain relief. In the present study, we have investigated the role of VEGF-A and its receptors, VEGFR-1 and VEGFR-2, in pain signalling and in chemotherapy-induced neuropathy establishment as well as the therapeutic potential of receptor blockade in the management of pain. Methods Behavioural and electrophysiological analyses were performed in an in vivo murine model, by using selective receptor agonists, blocking monoclonal antibodies or siRNA-mediated silencing of VEGF-A and VEGFRs. Expression of VEGF-A and VEGFR-1 in astrocytes and neurons was detected by immunofluorescence staining and confocal microscopy analysis. Results In mice, the intrathecal infusion of VEGF-A (VEGF(165) isoforms) induced a dose-dependent noxious hypersensitivity and this effect was mediated by VEGFR-1. Consistently, electrophysiological studies indicated that VEGF-A strongly stimulated the spinal nociceptive neurons activity through VEGFR-1. In the dorsal horn of the spinal cord of animals affected by oxaliplatin-induced neuropathy, VEGF-A expression was increased in astrocytes while VEGFR-1 was mainly detected in neurons, suggesting a VEGF-A/VEGFR-1-mediated astrocyte-neuron cross-talk in neuropathic pain pathophysiology. Accordingly, the selective knockdown of astrocytic VEGF-A by intraspinal injection of shRNAmir blocked the development of oxaliplatin-induced neuropathic hyperalgesia and allodynia. Interestingly, both intrathecal and systemic administration of the novel anti-VEGFR-1 monoclonal antibody D16F7, endowed with anti-angiogenic and antitumor properties, reverted oxaliplatin-induced neuropathic pain. Besides, D16F7 effectively relieved hypersensitivity induced by other neurotoxic chemotherapeutic agents, such as paclitaxel and vincristine. Conclusions These data strongly support the role of the VEGF-A/VEGFR-1 system in mediating chemotherapy-induced neuropathic pain at the central nervous system level. Thus, treatment with the anti-VEGFR-1 mAb D16F7, besides exerting antitumor activity, might result in the additional advantage of attenuating neuropathic pain when combined with neurotoxic anticancer agents