mTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1.

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.

A Dual-Acting Nitric Oxide Donor and Phosphodiesterase 5 Inhibitor Activates Autophagy in Primary Skin Fibroblasts.

Wound healing pathologies are an increasing problem in ageing societies. Chronic, non-healing wounds, which cause high morbidity and severely reduce the quality of life of affected individuals, are frequently observed in aged individuals and people suffering from diseases affected by the Western lifestyle, such as diabetes. Causal treatments that support proper wound healing are still scarce. Here, we performed expression proteomics to study the effects of the small molecule TOP-N53 on primary human skin fibroblasts and keratinocytes. TOP-N53 is a dual-acting nitric oxide donor and phosphodiesterase-5 inhibitor increasing cGMP levels to support proper wound healing. In contrast to keratinocytes, which did not exhibit global proteome alterations, TOP-N53 had profound effects on the proteome of skin fibroblasts. In fibroblasts, TOP-N53 activated the cytoprotective, lysosomal degradation pathway autophagy and induced the expression of the selective autophagy receptor p62/SQSTM1. Thus, activation of autophagy might in part be responsible for beneficial effects of TOP-N53.

Increased abundance of Cbl E3 ligases alters PDGFR signaling in recessive dystrophic epidermolysis bullosa.

In recessive dystrophic epidermolysis bullosa (RDEB), loss of collagen VII, the main component of anchoring fibrils critical for epidermal-dermal cohesion, affects several intracellular signaling pathways and leads to impaired wound healing and fibrosis. In skin fibroblasts, wound healing is also affected by platelet-derived growth factor receptor (PDGFR) signaling. To study a potential effect of loss of collagen VII on PDGFR signaling we performed unbiased disease phosphoproteomics. Whereas RDEB fibroblasts exhibited an overall weaker response to PDGF, Cbl E3 ubiquitin ligases, negative regulators of growth factor signaling, were stronger phosphorylated. This increase in phosphorylation was linked to higher Cbl mRNA and protein levels due to increased TGFß signaling in RDEB. In turn, increased Cbl levels led to increased PDGFR ubiquitination, internalization, and degradation negatively affecting MAPK and AKT downstream signaling pathways. Thus, our results indicate that elevated TGFß signaling leads to an attenuated response to growth factors, which contributes to impaired dermal wound healing in RDEB.

ULK1 phosphorylation of striatin activates protein phosphatase 2A and autophagy.

The evolutionarily conserved ULK1 kinase complex acts as gatekeeper of canonical autophagy and regulates induction of autophagosome biogenesis. To better understand control of ULK1 and analyze whether ULK1 has broader functions that are also linked to the later steps of autophagy, we perform comprehensive phosphoproteomic analyses. Combining in vivo with in vitro data, we identify numerous direct ULK1 target sites within autophagy-relevant proteins that are critical for autophagosome maturation and turnover. In addition, we highlight an intimate crosstalk between ULK1 and several phosphatase complexes. ULK1 is not only a PP2A target but also directly phosphorylates the regulatory PP2A subunit striatin, activating PP2A and serving as positive feedback to promote autophagy-dependent protein turnover. Thus, ULK1 and phosphatase activities are tightly coordinated to robustly regulate protein degradation by autophagy.

Effects of anti-PD-1 immunotherapy on tumor regression: insights from a patient-derived xenograft model.

Immunotherapies, such as checkpoint blockade of programmed cell death protein-1 (PD-1), have resulted in unprecedented improvements in survival for patients with lung cancer. Nonetheless, not all patients benefit equally and many issues remain unresolved, including the mechanisms of action and the possible effector function of immune cells from non-lymphoid lineages. The purpose of this study was to investigate whether anti-PD-1 immunotherapy acts on malignant tumor cells through mechanisms beyond those related to T lymphocyte involvement. We used a murine patient-derived xenograft (PDX) model of early-stage non-small cell lung carcinoma (NSCLC) devoid of host lymphoid cells, and studied the tumor and immune non-lymphoid responses to immunotherapy with anti-PD-1 alone or in combination with standard chemotherapy (cisplatin). An antitumor effect was observed in animals that received anti-PD-1 treatment, alone or in combination with cisplatin, likely due to a mechanism independent of T lymphocytes. Indeed, anti-PD-1 treatment induced myeloid cell mobilization to the tumor concomitant with the production of exudates compatible with an acute inflammatory reaction mediated by murine polymorphonuclear leukocytes, specifically neutrophils. Thus, while keeping in mind that more research is needed to corroborate our findings, we report preliminary evidence for a previously undescribed immunotherapy mechanism in this model, suggesting a potential cytotoxic action of neutrophils as PD-1 inhibitor effector cells responsible for tumor regression by necrotic extension.

Benefits of exercise and immunotherapy in a murine model of human non-small-cell lung carcinoma.

BACKGROUND: Lung cancer has the highest incidence and mortality rate in the world. One of the most promising new cancer therapies in recent years is immunotherapy, which is based on the blockade of immune checkpoints such as programmed cell death protein 1 (PD-1). Exercise training is beneficial to maintain and improve the quality of life of cancer patients, and it might also modulate the anti-tumoral efficiency of some chemotherapeutic agents. However, the potential of exercise combined with immunotherapy as a cancer therapy remains to be elucidated. Here, we examined the effects of exercise on tumor growth and its possible adjuvant effects when combined with anti-PD-1 immunotherapy (nivolumab) in a patient derived xenograft (PDX) model of non-small-cell lung carcinoma (NSCLC). METHODS: We generated a PDX model using NOD-SCID gamma mice with subcutaneous grafts from tumor tissue of a patient with NSCLC. Animals were randomly assigned to one of four groups: non-exercise + isotype control (n=5), exercise + isotype control (n=5), non-exercise + nivolumab (n=6) or exercise + nivolumab (n=6). The animals undertook an 8- week moderate-intensity training regimen (treadmill aerobic exercise and strength training). Immunotherapy (nivolumab) or an isotype control was administered 2 days/week, for 6 weeks. Several tumor growth and microenvironment parameters were measured after the intervention. RESULTS: Improvements in aerobic capacity and muscle strength (p=0.027 and p=0.005) were noted in exercised animals. Exercise alone reduced the tumor growth rate with respect to non-exercised mice (p=0.050). The double intervention (exercise + nivolumab) increased tumor necrosis and reduced apoptosis with respect to controls (p=0.026; p=0.030). All interventions achieved a reduction in proliferation compared with the control group (p=0.015, p=0.011, and p=0.011). Exercise alone increased myeloid tumor infiltrates (mostly neutrophils) with respect to the nivolumab only group (p=0.018). Finally, Vegf-a expression was higher in the nivolumab groups (in combination or not with exercise) than in exercise + isotype control group (p=0.045 and p=0.047, respectively). No other significant effects were found. CONCLUSIONS: Our results would suggest that aerobic and strength training should be studied as an adjuvant to cancer immunotherapy treatment.

CB2 cannabinoid receptor activation promotes colon cancer progression via AKT/GSK3ß signaling pathway.

The pharmacological activation of the cannabinoid receptor type 2, CB2, has been shown to elicit anti-tumoral mechanisms in different cancer types. However, little is known about its endogenous role in tumor pathophysiology, and different studies have attributed pro-tumorigenic properties to this receptor. In a previous work, we showed that CB2 expression is a poor prognostic factor in colon cancer patients. Here we report that activation of CB2 with low doses of specific agonists induce cell proliferation and favor the acquisition of aggressive molecular features in colon cancer cells. We show that sub-micromolar concentrations of CB2-specific agonists, JWH-133 and HU-308, promote an increase in cell proliferation rate through the activation of AKT/PKB pathway in colon cancer in vitro and in vivo. AKT activation promotes GSK3ß inhibition and thus, a more aggressive cell phenotype with the subsequent elevation of SNAIL levels, E-cadherin degradation and ß-catenin delocalization from cell membrane. Taken together, our data show that CB2 activation with sub-micromolar doses of agonists, which could be more similar to endogenous levels of cannabinoids, promote colon cancer progression, implicating that CB2 could have a pro-tumorigenic endogenous role in colon cancer.

Spontaneous Cannabinoid Receptor 2 (CB2) Expression in the Cochlea of Adult Albino Rat and Its Up-Regulation after Cisplatin Treatment.

We provide evidence for the presence of cannabinoid CB2 receptors in some cellular types of the cochlea of the adult albino rat. Cannabinoids and their receptors are increasingly being studied because of their high potential for clinical use. As a hyperspecialized portion of the peripheral nervous system, study of the expression and function of cannabinoid receptors in the hearing organ is of high interest. Stria vascularis and inner hair cells express CB2 receptor, as well as neurites and cell bodies of the spiral ganglion. Cellular types such as supporting cells and outer hair cells, in which the expression of other types of functional receptors has been reported, do not significantly express CB2 receptors in this study. An up-regulation of CB2 gene expression was detected after an ototoxic event such as cisplatin treatment, probably due to pro-inflammatory events triggered by the drug. That fact suggests promising potential of CB2 receptor as a therapeutic target for new treatments to palliate cisplatin-induced hearing loss and other ototoxic events which triggers inflammatory pathways.

Cannabinoids receptor type 2, CB2, expression correlates with human colon cancer progression and predicts patient survival.

Many studies have demonstrated that the endocannabinoid system (ECS) is altered in different tumor types, including colon cancer. However, little is known about the role of the ECS in tumor progression. Here we report the correlation between CB 2 expression and pathological data in a series of 175 colorectal cancer patients, as well as the response of the HT29 colon cancer-derived cell line upon CB 2 activation. CB 2 mRNA was detected in 28.6% of samples tested. It was more frequent in N+ patients and predicts disease free survival and overall survival in colon cancer. In positive samples, CB 2 was expressed with great intensity in tumor epithelial cells and correlated with tumor growth. Treatment of HT29 with CB 2 agonist revealed membrane loss of E-cadherin and SNAIL1 overexpression. A direct correlation between CB 2 and SNAIL1 expression was also found in human tumors. CB 2 receptor expression is a poor prognostic marker for colon cancer and the activation of this receptor, with non-apoptotic doses of agonists, could be collaborating with disease progression. These results raise the question whether the activation of CB 2 should be considered as anti-tumoral therapy.

Targeting multiple cannabinoid anti-tumour pathways with a resorcinol derivative leads to inhibition of advanced stages of breast cancer.

BACKGROUND AND PURPOSE: The psychoactive cannabinoid Δ(9) -tetrahydrocannabinol (THC) and the non-psychoactive cannabinoid cannabidiol (CBD) can both reduce cancer progression, each through distinct anti-tumour pathways. Our goal was to discover a compound that could efficiently target both cannabinoid anti-tumour pathways. EXPERIMENTAL APPROACH: To measure breast cancer cell proliferation/viability and invasion, MTT and Boyden chamber assays were used. Modulation of reactive oxygen species (ROS) and apoptosis was measured using dichlorodihydrofluorescein and annexin/propidium iodide, respectively, in combination with cell flow cytometry. Changes in protein levels were evaluated using Western analysis. Orthotopic and i.v. mouse models of breast cancer metastasis were used to test the activity of cannabinoids in vivo. KEY RESULTS: CBD reduced breast cancer metastasis in advanced stages of the disease as the direct result of down-regulating the transcriptional regulator Id1. However, this was associated with moderate increases in survival. We therefore screened for analogues that could co-target cannabinoid anti-tumour pathways (CBD- and THC-associated) and discovered the compound O-1663. This analogue inhibited Id1, produced a marked stimulation of ROS, up-regulated autophagy and induced apoptosis. Of all the compounds tested, it was the most potent at inhibiting breast cancer cell proliferation and invasion in culture and metastasis in vivo. CONCLUSIONS AND IMPLICATIONS: O-1663 prolonged survival in advanced stages of breast cancer metastasis. Developing compounds that can simultaneously target multiple cannabinoid anti-tumour pathways efficiently may provide a novel approach for the treatment of patients with metastatic breast cancer.

Gene expression profiling identifies EPHB4 as a potential predictive biomarker in colorectal cancer patients treated with bevacizumab.

The anti-VEGF monoclonal antibody bevacizumab was approved in 2004 as a first-line treatment for metastatic colorectal cancer (CRC) in combination with chemotherapy and provided proof of principle for antiangiogenic therapy. However, there is no biomarker that can help to select patients who may benefit from bevacizumab in order to improve cost-effectiveness and therapeutic outcomes. The aim of this study was to compare gene expression profiles in CRC patients treated with bevacizumab who responded to the treatment with those that did not respond, in an effort to identify potential predictive biomarkers. RNA isolated from formalin-fixed paraffin-embedded tumor specimens of patients treated with bevacizumab was subjected to gene expression analysis with quantitative RT-PCR arrays profiling 84 genes implicated in the angiogenic process. Data were validated at the protein level using immunohistochemistry. We identified a gene, EPHB4, whose expression was significantly increased in nonresponders (p = 0.048, Mann-Whitney test). Furthermore, high EPHB4 tumor levels were associated with decreased median overall survival (16 months vs 48, Log-rank p = 0.012). This was not observed in a control group of CRC patients treated only with chemotherapy, suggesting that EPHB4 constitutes a potential predictive biomarker and not a mere prognostic one. These data support the notion of a potential synergy between EPHB4-EFNB2 and VEGF-VEGFR pathways, making patients with high EPHB4 expression more resistant to VEGF blocking. Therefore, determination of EPHB4 levels in CRC samples could be useful for the prediction of response to bevacizumab.