Galectin-3 and cancer immunotherapy: a glycobiological rationale to overcome tumor immune escape.

Immunotherapy with checkpoint inhibitors (ICIs) has radically changed the landscape of therapeutic opportunities in oncology, but much still needs to be understood from a mechanistic point of view. There is space for further improving tumors' response to ICIs, as supported by a strong biological rationale. For this achievement a detailed analysis of tumor cell phenotype with functional dissection of the molecular interactions occurring in the TME is required. Galectin-3 is a pleiotropic tumor relevant molecule, which deserves particular attention in immuno-oncology. Due to its ability to finely modulate immune response in vivo, Galectin-3 is a potential target molecule to be considered for overcoming tumor immune escape.

Activated MKK3/MYC crosstalk impairs dabrafenib response in BRAFV600E colorectal cancer leading to resistance.

Colorectal cancer (CRC) patients with BRAF mutations develop resistance to BRAF inhibitors at a very early stage. Understanding the molecular mechanisms involved in BRAF inhibitor resistance is critical for the development of novel therapeutic opportunities for this subtype of CRC patients. CRC cells bearing BRAF mutations are mostly sensitive to the abrogation of Mitogen-Activated Protein Kinase Kinase 3 (MKK3), a specific activator of p38MAPKs signaling, suggesting that BRAF alterations might addict CRC cells to the MKK3/p38MAPK signaling. Interestingly, publicly available gene expression profiling data show significantly higher MKK3 transcript levels in CRC lines with acquired resistance to BRAF inhibitors. Herein, we investigated the roles of MKK3 in the response to BRAF targeting (dabrafenib) with COLO205 and HT29 BRAFV600E CRC lines and derived dabrafenib-resistant (DABR) sublines. Dabrafenib treatments reduce MKK3 activation by inducing autophagy in parental but not DABR cells. The MKK3 knockdown induces cell death in DABR cells, whereas ectopic MKK3 expression reduces dabrafenib sensitivity in parental cells. Mechanistically, activated MKK3 interacts and co-localizes with c-Myc oncoprotein (MYC), sustaining MYC protein stability and thus preventing the dabrafenib induced effects in CRC DABR cells both in vitro and in vivo. Overall, we identify a novel molecular mechanism beyond the dabrafenib resistance, shedding light on an uncovered vulnerability for the development of novel therapeutic opportunities in BRAFV600E CRC.

A combination of PARP and CHK1 inhibitors efficiently antagonizes MYCN-driven tumors.

MYCN drives aggressive behavior and refractoriness to chemotherapy, in several tumors. Since MYCN inactivation in clinical settings is not achievable, alternative vulnerabilities of MYCN-driven tumors need to be explored to identify more effective and less toxic therapies. We previously demonstrated that PARP inhibitors enhance MYCN-induced replication stress and promote mitotic catastrophe, counteracted by CHK1. Here, we showed that PARP and CHK1 inhibitors synergized to induce death in neuroblastoma cells and in primary cultures of SHH-dependent medulloblastoma, their combination being more effective in MYCN amplified and MYCN overexpressing cells compared to MYCN non-amplified cells. Although the MYCN amplified IMR-32 cell line carrying the p.Val2716Ala ATM mutation showed the highest sensitivity to the drug combination, this was not related to ATM status, as indicated by CRISPR/Cas9-based correction of the mutation. Suboptimal doses of the CHK1 inhibitor MK-8776 plus the PARP inhibitor olaparib led to a MYCN-dependent accumulation of DNA damage and cell death in vitro and significantly reduced the growth of four in vivo models of MYCN-driven tumors, without major toxicities. Our data highlight the combination of PARP and CHK1 inhibitors as a new potential chemo-free strategy to treat MYCN-driven tumors, which might be promptly translated into clinical trials.

May Bacterial Infections Trigger Bullous Pemphigoid? Case Report and Review of Literature.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease, mainly observed in the elderly. Infections have been suggested as possible disease triggers. However, infections may even heavily influence the disease clinical course and mortality. A 75-year-old woman was admitted to hospital for severe erythematosus blistering disease, accompanied by hyper-eosinophilia and hyper-IgE. The culture of bullous fluid was positive for Enterococcus faecalis, the blood culture was positive for Staphylococcus aureus, and the urine culture was positive for Proteus mirabilis and Escherichia coli. Moreover, circulating anti-BP180 IgG was present and the histopathological/ultrastructural examination of a lesional skin biopsy was compatible with BP. High eosinophil levels (up to 3170/µL) were found throughout the clinical course, while values below 1000/µL were associated with clinical improvement. The total IgE was 1273 IU/mL, and specific anti-G/V-penicillin/ampicillin IgE antibodies were positive. The patient had a complete clinical recovery in two months with methyl-prednisolone (40 then 20 mg/day) and low-dose azathioprine (50 mg/day) as a steroid-sparing agent. The steroid treatment was tapered until interruption during a one-year period and intravenous immunoglobulins have been administered for three years in order for azathioprine to also be interrupted. The patient stopped any treatment five years ago and, in this period, has always been in good health. In this case, the contemporaneous onset of different bacterial infections and BP is suggestive of bacterial infections acting as BP trigger(s), with allergic and autoimmune pathways contributing to the disease pathogenesis.

In Vivo Imaging of Thyroid Cancer with 99mTc-TR1401 and 99mTc-TR1402: A Comparison Study in Dogs.

Differentiated thyroid cancer (DTC) cells may lose NIS expression and iodine uptake, but usually express TSH receptors (TSHR). Therefore, the aim of our study was to compare two radiolabeled superagonist TSH analogues for DTC imaging. These analogues (namely TR1401 and TR1402) have a higher TSHR binding affinity than recombinant human TSH (Thyrogen®). Radiolabeling was performed with technetium-99m using an indirect method via HYNIC conjugation and was followed by in vitro quality controls and binding assay on TSHR-positive cell lines (ML-1). An in vitro binding assay was also performed and compared with radiolabeled human recombinant TSH. In vivo imaging was performed in four dogs with spontaneous follicular thyroid carcinoma with solid poorly differentiated areas with 99mTc-TR1401 SPECT/CT, 99mTc-TR1402 SPECT/CT, and [18F]FDG PET/CT on different days within 2 weeks. TR1401 and TR1402 were labeled with high specific activity (8.3 ± 1.2 MBq/µg) and retention of their biological activity and structural integrity. Both agonists were able to efficiently bind TSHR receptors expressed by cell lines with dissociation constants (Kd) of 2.7 nM for 99mTc-TR1401 and 0.5 nM for 99mTc-TR1402 compared with 99mTc-Thyrogen (Kd = 8.4 nM). In tumor-targeting experiments, a focal uptake was observed in dogs with spontaneous intraglandular thyroid carcinoma, in which TSHR expression was confirmed by immunohistochemistry. 99mTc-TR1402 provided higher T/B than 99mTc-TR1401 and [18F]FDG (12.9 ± 1.3, 10.2 ± 0.7, and 3.8 ± 0.6, respectively; all p < 0.001). Given these results, 99mTc-TR1402 appears to be a useful tool for in vivo imaging of thyroid cancer.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo.

The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin VH/VL regions of which one-employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66-showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (KD = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.

TP53 drives abscopal effect by secretion of senescence-associated molecular signals in non-small cell lung cancer.

BACKGROUND: Recent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. However, deeper understanding of the molecular mechanisms underlying the abscopal effect are required to best benefit a larger proportion of patients with metastasis. Several groups including ours, reported the involvement of wild-type (wt) p53 in radiation-induced abscopal effects, however very little is known on the role of wtp53 dependent molecular mechanisms. METHODS: We investigated through in vivo and in vitro approaches how wtp53 orchestrates radiation-induced abscopal effects. Wtp53 bearing (A549) and p53-null (H1299) NSCLC lines were xenotransplanted in nude mice, and cultured in 2D monolayers and 3D tumor spheroids. Extracellular vesicles (EVs) were isolated from medium cell culture by ultracentrifugation protocol followed by Nanoparticle Tracking Analysis. Gene expression was evaluated by RT-Real Time, digital qRT-PCR, and dot blot technique. Protein levels were determined by immunohistochemistry, confocal anlysis, western blot techniques, and immunoassay. RESULTS: We demonstrated that single high-dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 xenograft tumors but not in NIR p53-null H1299 or p53-silenced A549 (A549sh/p53) xenografts. We further demonstrates that irradiation of A549 cells in vitro induces a senescence-associated secretory phenotype (SASP) producing extracellular vesicles (EVs) expressing CD63 and carrying DNA:RNA hybrids and LINE-1 retrotransposon. IR-A549 EVs also hamper the colony-forming capability of recipient NIR A549 cells, induce senescent phenotype, nuclear expression of DNA:RNA hybrids, and M1 macrophage polarization. CONCLUSIONS: In our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of SASP and secretion of CD63+ EVs loaded with DNA:RNA hybrids and LINE-1 retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors.

Imaging atherosclerotic plaques by targeting Galectin-3 and activated macrophages using (89Zr)-DFO- Galectin3-F(ab')2 mAb.

Rationale: The high expression of Galectin-3 (Gal3) in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity in vivo. Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice via PET imaging. Methods: Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed in vitro using a Gal3-F(ab')2 mAb labeled with AlexaFluor®488 and 89Zr- desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO). To visualize plaques in vivo, ApoE-KO mice were injected i.v. with 89Zr-DFO-Gal3-F(ab')2 mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab')2 mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3 (i.e. a synonym for Gal3) gene expression in each macrophage of several subtypes present in murine or human plaques. Results: Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab')2 and 89Zr-DFO-Gal3-F(ab')2 mAbs. Focal and specific 89Zr-DFO-Gal3-F(ab')2 mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts. Conclusions: Our data reveal that 89Zr-DFO-Gal3-F(ab')2 mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease.

Development of a Chimeric Antigen-Binding Fragment Directed Against Human Galectin-3 and Validation as an Immuno-Positron Emission Tomography Tracer for the Sensitive In Vivo Imaging of Thyroid Cancer.

Background: The lack of facile methods for the specific characterization of malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Due to its restricted expression in such nodules, the cell-associated lectin galectin-3 (Gal3) has emerged as a marker for TC with growing interest for in vivo imaging as well as targeted radionuclide therapy. To accelerate translation into clinical application, we have developed a cognate chimeric human antigen-binding fragment (Fab) derived from the rat anti-Gal3 monoclonal antibody M3/38. Methods: The variable immunoglobulin (Ig) light and heavy chain sequences were cloned from the hybridoma cell line, and the corresponding Fab carrying human IgG1/κ constant genes was functionally produced in the periplasm of Escherichia coli and purified to homogeneity. To moderately prolong its plasma half-life and, thus, increase tumor uptake, the recombinant Fab was fused with a long disordered amino acid chain comprising in total 200 Pro, Ala, and Ser residues (PASylation). This novel tracer was subjected to in vitro characterization and in vivo validation by using two thyroid cancer orthotopic murine models. To this end, the αGal3-Fab-PAS200 was conjugated with deferoxamine (Dfo), labeled with 89Zr under mild conditions and tested for binding on TC cell lines. Athymic nude mice were inoculated either with FRO82-1 or with CAL62 tumor cells into the left thyroid lobe. After intravenous injection with ∼3.0 MBq of 89Zr-Dfo-PAS200-Fab, these mice were subjected to positron emission tomography (PET)/computed tomography imaging followed by quantification of tumor accumulation and immunohistochemical analysis. Results: The αGal3-Fab-PAS200 revealed high affinity toward the recombinant Gal3 antigen, with a dissociation constant ≤1 nM as measured via enzyme-linked immunosorbent assay, surface plasmon resonance spectroscopy, and radioactive cell binding assay. The in vivo Gal3-targeting by the 89Zr(IV)-labeled protein tracer, as investigated by immuno-PET, demonstrated highly selective and fast accumulation in orthotopically implanted tumors, with strong contrast images achieved 24 hours postinjection, and no uptake in the tumor-free thyroid lobe, as also confirmed by biodistribution studies. Conclusions: The chimeric αGal3 89Zr-Dfo-PAS200-Fab tracer exhibits selective accumulation in the tumor-bearing thyroid lobe of xenograft mice. Thus, this novel radioactive probe offers potential to change TC management, in addition to current diagnostic procedures, and to reduce unnecessary thyroidectomies.

MKK3 sustains cell proliferation and survival through p38DELTA MAPK activation in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and understanding its underlying molecular mechanisms is crucial for the development of therapeutic strategies. The mitogen-activated protein kinase-kinase 3 (MKK3) is a specific activator of p38 MAP kinases (p38 MAPKs), which contributes to the regulation of several cellular functions, such as proliferation, differentiation, apoptosis as well as response to drugs. At present, the exact MKK3/p38 MAPK pathway contribution in cancer is heavily debated because of its pleiotropic function. In this work, we retrospectively explored the prognostic and pathobiologic relevance of MKK3 in a cohort of CRC patients and assessed MKK3 molecular functions in a panel of CRC lines and colonocytes primary cultures. We found increased MKK3 levels in late-stage CRC patients which correlated with shorter overall survival. Herein, we report that the MKK3 targeting by inducible RNA interference univocally exerts antitumor effects in CRC lines but not in primary colonocytes. While MKK3 depletion per se affects growth and survival by induction of sustained autophagy and death in some CRC lines, it potentiates response to chemotherapeutic drug 5-fluorouracil (5-FU) in all of the tested CRC lines in vitro. Here, we demonstrate for the first time that in CRC the MKK3 specifically activates p38delta MAPK isoform to sustain prosurvival signaling and that such effect is exacerbated upon 5-FU challenge. Indeed, p38delta MAPK silencing recapitulates MKK3 depletion effects in CRC cells in vitro and in vivo. Overall, our data identified a molecular mechanism through which MKK3 supports proliferation and survival signaling in CRC, further supporting MKK3 as a novel and extremely attractive therapeutic target for the development of promising strategies for the management of CRC patients.

Predictive Biomarkers for Checkpoint Inhibitor-Based Immunotherapy: The Galectin-3 Signature in NSCLCs.

Checkpoint inhibitor-based immunotherapy is opening a promising scenario in oncology, with objective responses registered in multiple cancer types. However, reliable predictive markers of tumor responsiveness are still lacking. These markers need to be urgently identified for a better selection of patients that can be candidates for immunotherapy. In this pilot study, a cohort of 34 consecutive patients bearing programmed death-ligand 1 (PD-L1)-positive non-small cell lung carcinoma (NSCLC), treated with pembrolizumab, was considered. The retrospective immuno-phenotypic analysis performed on the original tumor biopsies allowed for the identification of a specific "galectin signature", which strongly correlated with tumor responsiveness to anti PD-1 immunotherapy. We observed that the large majority of patients (about 90%) with high galectin-3 tumor expression (score 3+) showed an early and dramatic progression of the disease after three cycles of treatments. In contrast, all patients with negative or low/intermediate expression of galectin-3 in tumor cells showed an early and durable objective response to pembrolizumab, indicating galectin-3 as an interesting predictive marker of tumor responsiveness. The galectin-3 signature, at least in NSCLCs, promises a better selection of patient candidates for immunotherapy, reducing unnecessary treatment exposures and social costs. A large multicenter study is ongoing to validate this finding.

25-Hydroxyvitamin D serum levels and melanoma risk: a case-control study and evidence synthesis of clinical epidemiological studies.

There is accumulating evidence that the vitamin D pathway may play a role in melanoma. The aim of this study was to investigate the association between 25-hydroxyvitamin D [25(OH)D] serum levels and the risk of cutaneous melanoma. A case-control study with 137 incident cases of melanoma (serum samples collected at the time of diagnosis) and 99 healthy controls (serum samples collected between October and April) was carried out and evaluated in the framework of an evidence synthesis of clinical epidemiological studies on the topic to facilitate comparisons and summarize the scientific evidence produced so far. There was a statistically significant difference in the median levels of serum vitamin D between melanoma patients and healthy controls (18.0 vs. 27.8 ng/ml, P<0.001). Among melanoma patients, 66.2%, compared with 15.2% of healthy controls, had vitamin D deficiency (≤20 ng/ml), whereas vitamin D sufficiency (≥30 ng/ml) was observed in only 7.4% of melanoma patients and in 37.4% of the healthy controls (P<0.001). A multivariate model including age, sex, and BMI showed a statistically significant inverse association between melanoma and vitamin D sufficiency versus deficiency (odds ratio=0.04; 95% confidence interval: 0.02-0.10, P<0.001). Also, vitamin D insufficiency versus deficiency was significantly inversely associated with melanoma (odds ratio=0.13; 95% confidence interval: 0.06-0.27, P<0.001). These results suggest that both deficient and insufficient serum levels of vitamin D are associated with melanoma and that a trend seems to be present with a reduced risk of melanoma when vitamin D approaches normal values.

A "seasonal bromoderma" in a farmer.

Bromoderma is a rare skin disease caused by the ingestion, inhalation, or contact with products containing bromides. We report a case of bromoderma in a 36-year-old farmer, characterized by remission during the spring and exacerbation during the winter. The recognition of the use of products containing bromides during the winter helped us to understand the cause of the clinical condition.

Galectin-3 Targeting in Thyroid Orthotopic Tumors Opens New Ways to Characterize Thyroid Cancer.

Preoperative characterization of thyroid nodules is challenging since thyroid scintigraphy fails to distinguish between benign and malignant lesions. Galectin-3 (gal-3) is expressed in well-differentiated and in undifferentiated thyroid cancer types but not in normal thyrocytes and benign thyroid lesions. Herein, we aimed to validate gal-3 targeting as a specific method to detect non-radioiodine-avid thyroid cancer in thyroid orthotopic tumor models. Methods: Papillary (BcPAP) and anaplastic (CAL62 and FRO82-1) thyroid carcinoma cell lines were characterized via Western blot and polymerase chain reaction for gal-3 and sodium-iodide symporter (NIS) expression. An 89Zr-labeled F(ab')2 antigal-3 was generated and characterized for binding versus 125I on 2- and 3-dimensional cell cultures. The thyroid carcinoma cells were inoculated into the left thyroid lobe of athymic nude mice, and the orthotopic tumor growth was monitored via ultrasound and fluorescence molecular tomography. Head-to-head PET/CT comparison of 124I versus 89Zr-deferoxamine (DFO)-F(ab')2 antigal-3 was performed, followed by biodistribution studies and immunohistochemical analysis for gal-3 and NIS expression. Results: The thyroid carcinoma cells investigated were invariably gal-3-positive while presenting low or lost NIS expression. 89Zr-DFO-F(ab')2 antigal-3 tracer showed high affinity to gal-3 (dissociation constant, ∼3.9 nM) and retained immunoreactivity (>75%) on 2-dimensional cell cultures and on tumor spheroids. 125I internalization in FRO82-1, BcPAP, and CAL62 was directly dependent on NIS expression, both in 2-dimensional and tumor spheroids. PET/CT imaging showed 89Zr-DFO-F(ab')2 antigal-3 signal associated with the orthotopically implanted tumors only; no signal was detected in the tumor-free thyroid lobe. Conversely, PET imaging using 124I showed background accumulation in tumor-infiltrated lobe, a condition simulating the presence of non-radioiodine-avid thyroid cancer nodules, and high accumulation in normal thyroid lobe. Imaging data were confirmed by tracer biodistribution studies and immunohistochemistry. Conclusion: A specific and selective visualization of thyroid tumor by targeting gal-3 was demonstrated in the absence of radioiodine uptake. Translation of this method into the clinical setting promises to improve the management of patients by avoiding the use of unspecific imaging methodologies and reducing unnecessary thyroid surgery.

Inhibition of Stearoyl-CoA desaturase 1 reverts BRAF and MEK inhibition-induced selection of cancer stem cells in BRAF-mutated melanoma.

BACKGROUND: Combination therapy with BRAF and MEK inhibitors significantly improves survival in BRAF mutated melanoma patients but is unable to prevent disease recurrence due to the emergence of drug resistance. Cancer stem cells (CSCs) have been involved in these long-term treatment failures. We previously reported in lung cancer that CSCs maintenance is due to altered lipid metabolism and dependent upon Stearoyl-CoA-desaturase (SCD1)-mediated upregulation of YAP and TAZ. On this ground, we investigated the role of SCD1 in melanoma CSCs. METHODS: SCD1 gene expression data of melanoma patients were downloaded from TCGA and correlated with disease progression by bioinformatics analysis and confirmed on patient's tissues by qRT-PCR and IHC analyses. The effects of combination of BRAF/MEKi and the SCD1 inhibitor MF-438 were monitored by spheroid-forming and proliferation assays on a panel of BRAF-mutated melanoma cell lines grown in 3D and 2D conditions, respectively. SCD1, YAP/TAZ and stemness markers were evaluated in melanoma cells and tissues by qRT-PCR, WB and Immunofluorescence. RESULTS: We first observed that SCD1 expression increases during melanoma progression. BRAF-mutated melanoma 3D cultures enriched for CSCs overexpressed SCD1 and were more resistant than 2D differentiated cultures to BRAF and MEK inhibitors. We next showed that exposure of BRAF-mutated melanoma cells to MAPK pathway inhibitors enhanced stemness features by upregulating the expression of YAP/TAZ and downstream genes but surprisingly not SCD1. However, SCD1 pharmacological inhibition was able to downregulate YAP/TAZ and to revert at the same time CSC enrichment and resistance to MAPK inhibitors. CONCLUSIONS: Our data underscore the role of SCD1 as prognostic marker in melanoma and promote the use of SCD1 inhibitors in combination with MAPK inhibitors for the control of drug resistance.