The immunotherapy with immune checkpoints inhibitors (ICI) has changed the life expectancy in metastatic melanoma (MM) patients. Nevertheless, several patients do not respond hence, the ...identification and validation of novel biomarkers of response to ICI is of crucial importance. Circulating extracellular vesicles (EVs) such as PD-L1
EV mediate resistance to anti-PD1, instead the role of PD1
EV is not fully understood.
We isolated the circulating EVs from the plasma of an observational cohort study of 71 metastatic melanoma patients and correlated the amount of PD-L1
EVs and PD1
EVs with the response to ICI. The analysis was performed according to the origin of EVs from the tumor and the immune cells. Subsequently, we analysed the data in a validation cohort of 22 MM patients to assess the reliability of identified EV-based biomarkers. Additionally we assessed the involvement of PD1
EVs in the seizure of nivolumab and in the perturbation of immune cells-mediated killing of melanoma spheroids.
The level of PD-L1
EVs released from melanoma and CD8
T cells and that of PD1
EVs irrespective of the cellular origin were higher in non-responders. The Kaplan-Meier curves indicated that higher levels of PD1+ EVs were significantly correlated with poorer progression-free survival (PFS) and overall survival (OS). Significant correlations were found for PD-L1
EVs only when released from melanoma and T cells. The multivariate analysis showed that high level of PD1
EVs, from T cells and B cells, and high level of PD-L1
EVs from melanoma cells, are independent biomarkers of response. The reliability of PD-L1
EVs from melanoma and PD1
EVs from T cells in predicting PFS was confirmed in the validation cohort through the univariate Cox-hazard regression analysis. Moreover we discovered that the circulating EVs captured nivolumab and reduced the T cells trafficking and tumor spheroids killing.
Our study identified circulating PD1
EVs as driver of resistance to anti-PD1, and highlighted that the analysis of single EV population by liquid biopsy is a promising tool to stratify MM patients for immunotherapy.
In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin ...(Ln)‐332, produced by hepatic stellate cells (HSCs). Ln‐332 is the ligand of α3β1 and α6β4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln‐332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln‐332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC‐CM or Ln‐332 inhibited sorafenib's effectiveness in HCC cells expressing both α3β1 and α6β4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln‐332 and HSC‐CM. Hep3B cells expressing α6β4 but lacking the α3 integrin were insensitive to Ln‐332 and HSC‐CM protective effects. Hep3B α3‐positive, but not wild‐type and scramble transfected, cells acquired protection by sorafenib when plated on Ln‐332‐CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal‐regulated kinases 1/2, whereas Ln‐332 and HSC‐CM partially restored the pathways. Silencing FAK, but not extracellular signal‐regulated kinases 1/2, abrogated the protection induced by Ln‐332 and HSC‐CM, suggesting a specific role for FAK. Sorafenib down‐regulated total FAK, inducing its proteasomal degradation, while Ln‐332 and HSC‐CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. Conclusion: This study unveils a novel mechanism of sorafenib resistance depending on the α3β1/Ln‐332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103‐2117).
Standard chemotherapy for soft tissue sarcomas has shown limited efficacy. Here, we sought to evaluate whether β-adrenergic receptor (β-AR) signalling contributed to the progression of sarcomas and ...therapy resistance. To assess the translational potential of β-adrenergic receptors, we performed immunohistochemical detection of β1-AR, β2-AR and β3-AR in leiomyosarcoma, liposarcoma and angiosarcoma tissue specimens, reporting the results scored for the intensity. By using established and patient-derived sarcoma cells, we demonstrated the antitumour potential of the pharmacological targeting of β-ARs with the nonselective β-blocker propranolol in such sarcomas. Of note, pharmacological β-AR inhibition synergized with doxorubicin in inhibiting the cell viability of liposarcoma and leiomyosarcoma cells and increased the response to docetaxel in angiosarcoma- and solitary fibrous tumour (SFT)-patient-derived cells. Notably, the SFT patient was treated with the combination of propranolol and docetaxel, reporting prolonged disease control. Mechanistically, we found that propranolol reduced the activity of the multidrug resistance efflux pump P-gp, thereby increasing the intracellular doxorubicin concentration and antitumour activity. In addition, propranolol attenuated the Akt-dependent survival signal induced by doxorubicin and strongly reduced the activation of the NF-kB/COX-2 pathway, increasing cell sensitivity to docetaxel. Overall, our study highlighted the therapeutic potential of propranolol, alone or in rational combination therapies, for sarcoma treatment.
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Reported here is the synthesis and biological evaluation of the asialoglycoprotein receptor (ASGP-R) targeted fourth generation poliamidoamine dendrimer (G(4)-PAMAM) loaded with ...sorafenib. The ASGP-R targeted dendrimer was obtained by conjugation of Lactobionic acid (La) to the G(4)-PAMAM dendrimer, followed by acetylation (Ac) of the free amino groups in order to reduce the non-specific interactions with the cell membrane. Moreover, by additionally grafting fluorescein (FITC), it was easy to characterize the internalization pathway and the intracellular fate of the targeted dendrimer Ac-La-G(4)-PAMAM-FITC. In vitro experiments performed on HepG-2 and HLE cell lines, allowed to study the ability of the dendrimers to affect the cell vitality. Confocal microscopy and cytofluorimetric analysis confirmed higher binding and uptake ability of the Ac-La-G(4)-PAMAM-FITC dendrimer in well differentiated and ASGP-R expressing human liver cancer cell line HepG-2 compared non-expressing HLE cells. Ac-La-G(4)-PAMAM-FITC dendrimer loaded with sorafenib was stable and showed sustained sorafenib release. As evidenced by the cytotoxicity studies, sorafenib included in the dendrimer maintained its effectiveness, and was able to produce a longer lasting effect over the time compared to molar equivalent doses of free sorafenib. This new targeted dendrimer appears to be a suitable carrier for the delivery of sorafenib to liver cancer cells expressing ASGP-R.
Cutaneous squamous cell carcinoma (CSCC) is the most common keratinocyte-derived skin cancer in the Caucasian population. Exposure to UV radiations (UVRs) represents the main risk carcinogenesis, ...causing a considerable accumulation of DNA damage in epidermal keratinocytes with an uncontrolled hyperproliferation and tumor development. The limited and rarely durable response of CSCC to the current therapeutic options has led researchers to look for new therapeutic strategies. Recently, the multi-omics approaches have contributed to the identification and prediction of the key role of non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), circularRNAs (circRNAs) and long non-coding RNAs (lncRNAs) in the regulation of several cellular processes in different tumor types, including CSCC. ncRNAs can modulate transcriptional and post-transcriptional events by interacting either with each other or with DNA and proteins, such as transcription factors and RNA-binding proteins. In this review, the implication of ncRNAs in tumorigenesis and their potential role as diagnostic biomarkers and therapeutic targets in human CSCC are reported.
Tumor⁻stroma interactions are of key importance for pancreatic ductal adenocarcinoma (PDAC) progression. Our aim was to investigate whether cancer associated fibroblasts (CAFs) and mast cells (MC) ...affected the sensitivity of PDAC cells to gemcitabine/nabpaclitaxel (GEM/NAB). For this purpose, the combination cytotoxicity and the effect on tumor invasion and angiogenesis were evaluated with or without a conditioned medium from the mast cell line HMC-1 (human mast cell line-1 cells) and CAFs. Beside the clinical outcome of a homogenous population of PDAC patients, receiving GEM/NAB, was correlated to the circulating levels of mast cell tryptase and to a panel of inflammatory and immunosuppressive cytokines. CAFs neither affected drugs' cytotoxicity nor the inhibition of angiogenesis, but promoted tumor cell invasion. The MC instead, caused resistance to drugs by reducing apoptosis, by activating the TGF-β signalling and by promoting tumor invasion. Indeed, the inhibition of TβRI serine/threonine kinase activity by galunisertib restored drugs cytotoxicity. Moreover, MC induced the release of TGF-β1, and increased expression of PAR-2, ERK1/2 and Akt activation. Accordingly, TGF-β1, tryptase and other pro-inflammatory and immunosuppressive cytokines increased in the unresponsive patients. In conclusion, MC play a pivotal role in the resistance to GEM/NAB. A correlation between high level of circulating pro-inflammatory/ immunosuppressive cytokines and unresponsiveness was found in PDAC patients.
Over the past several years there has been much debate with regards to the prognostic and clinical significance of pancreatic ductal adenocarcinoma (PDAC) with lymph nodes metastasis. The PDAC gene ...expression knowledge and the biologic alterations underlying the lymph node involvement convey a clinical implication in dealing with the theranostic window. To this end, we provide an original bioinformatic dissection of the gene expression differences of PDAC according to the nodal involvement from a large public available dataset. Comprehensive transcriptomic analysis from 143 RNA-seq patient's derived samples indicated that WNT increased activation and a peculiar immune microenvironment identify subjects with nodal involvement. In frame of this thinking, we validated the WNT pathway role in increasing the likelihood of lymphatic dissemination in vitro. Moreover, we demonstrated for the first time in a PDAC model the potential therapeutic window that XAV-939-a specific WNT pathway inhibitor-has in re-educating a tumor-permissive immune system. Finally, we outline the potential implication on bystander molecular drivers exerted by WNT molecular inhibition, providing a picture of the proteomic oncogenic landscape changes elicited by XAV-939 on PDAC cells and their clinical implication. Our findings hold the promise to identify novel immune-based therapeutic strategies targeting WNT to enhance PDAC cytotoxicity and restore anti-PDAC immunity in node-positive disease.
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•Notch boosts MAPK signaling in mutated uveal and cutaneous melanoma.•Notch inhibition counteracts GNAQ-induced MAPK activation.•Galunisertib prolongs the efficacy of cobimetinib in ...GNAQQ209L UM cells.•Simultaneous inhibition of Notch and MEK induces senescence of GNAQQ209L UM cells.
The crosstalk between Notch and MAPK pathway plays a role in MEK inhibitor resistance in BRAFV600E metastatic melanoma (MM) and promotes migration in GNAQQ209L uveal melanoma (UM) cells. We determined the cytotoxicity of combinatorial inhibition of MEK and Notch by cobimetinib and γ-secretase inhibitor (GSI) nirogacestat, in BRAFV600E and BRAF wt MM and GNAQQ209L UM cells displaying different Erk1/2 and Notch activation status, with the aim to elucidate the impact of Notch signaling in the response to MEK inhibitor. Overall the combination was synergic in BRAFV600E MM and GNAQQ209L UM cells and antagonistic in BRAF wt one. Focusing on UM cells, we found that cobimetinib resulted in G0/G1 phase arrest and apoptosis induction, whereas the combination with GSI increased treatment efficacy by inducing a senescent-like state of cells and by blocking migration towards liver cancer cells. Mechanistically, this was reflected in a strong reduction of cyclin D1, in the inactivation of retinoblastoma protein and in the increase of p27KIP1 expression levels. Of note, each drug alone prevented Notch signaling activation resulting in inhibition of c-jun(Ser63) and Hes-1 expression. The combination achieved the strongest inhibition on Notch signaling and on both c-jun(Ser63) and Erk1/2 activation level. In conclusion we unveiled a coordinate action of MAPK and Notch signaling in promoting proliferation of BRAFV600E MM and GNAQQ209L UM cells. Remarkably, the simultaneous inhibition of MEK and Notch signaling highlighted a role for the second pathway in protecting cells against senescence in GNAQQ209L UM cells treated with the MEK inhibitor.
Melanoma and non-melanoma skin cancers (NMSCs) are the most frequent cancers of the skin in white populations. An increased risk in the development of skin cancers has been associated with the ...combination of several environmental factors (i.e., ultraviolet exposure) and genetic background, including melanocortin-1 receptor (
) status. In the last few years, advances in the diagnosis of skin cancers provided a great impact on clinical practice. Despite these advances, NMSCs are still the most common malignancy in humans and melanoma still shows a rising incidence and a poor prognosis when diagnosed at an advanced stage. Efforts are required to underlie the genetic and clinical heterogeneity of melanoma and NMSCs, leading to an optimization of the management of affected patients. The clinical implications of the impact of germline
variants in melanoma and NMSCs' risk, together with the additional risk conferred by somatic mutations in other peculiar genes, as well as the role of
screening in skin cancers' prevention will be addressed in the current review.
There is a growing interest in the cytotoxic effects of bioactive glycoalkaloids, such as α-tomatine on tumor cells. Here, for the first time, we determine the antitumor potential of tomatine, a ...mixture of α-tomatine and dehydrotomatine, in metastatic melanoma (MM) cell lines harboring different BRAF and MC1R variants. We performed cytotoxicity experiments and annexin-V/propidium iodide staining to assess the apoptotic/necrotic status of the cells. ER stress and autophagy markers were revealed by Western Blot, whereas antiangiogenic and vascular-disrupting effects were evaluated through a capillary tube formation assay on matrigel and by ELISA kit for VEGF release determination. Cell invasion was determined by a Boyden chamber matrigel assay. Tomatine reduced 50% of cell viability and induced a concentration-dependent increase of apoptotic cells in the range of 0.5-1 μM in terms of α-tomatine. The extent of apoptosis was more than two-fold higher in
BRAF-D184H/D184H MC1R cells than in BRAF wild-type cells and
BRAF-MC1R wild-type cell lines. Additionally, tomatine increased the LC3I/II autophagy marker, p-eIF2α, and p-Erk1/2 levels in BRAF wild-type cells. Notably, tomatine strongly reduced cell invasion and melanoma-dependent angiogenesis by reducing VEGF release and tumor-stimulating effects on capillary tube formation. Collectively, our findings support tomatine as a potential antitumor agent in MM.