Functional characterization of NTRK1 mutations identified in melanoma.

Cutaneous melanoma is the most aggressive form of skin cancer, with a complex and heterogeneous aetiology. Deregulation of the mitogen activated protein kinase cascade is common in melanoma, due to activating mutations in the BRAF and NRAS genes. Genetic studies and high-throughput screening technologies have recently identified several somatic mutations affecting different receptor tyrosine kinase (RTK) genes. For the majority of these, however, the contribution to the complexity of melanoma biology has not been assessed. Among these, two novel missense somatic mutations (M379I and R577G) have recently been identified in the gene encoding the neurotrophic RTK NTRK1. The NTRK1 melanoma-associated point mutations were introduced in a NTRK1 expression plasmid. Functional characterization of mutants was assessed after transient and stable transfection in HeLa and NIH3T3 cells, respectively. We showed that M379I and R577G NTRK1 receptors do not display the kinase as constitutively activated and are functionally indistinguishable from the wild-type NTRK1 receptor. Our results indicate that a causative role for M379I and R577G NTRK1 mutations in melanoma development is highly unlikely. This supports the issue that, in parallel to systematic large scale cancer genome screening, functional studies are required to distinguish between mutations that play a causative role in tumor development and others that may only be passenger changes.

Non-oncogene dependencies: Novel opportunities for cancer therapy.

Targeting oncogene addictions have changed the history of subsets of malignancies and continues to represent an excellent therapeutic opportunity. Nonetheless, alternative strategies are required to treat malignancies driven by undruggable oncogenes or loss of tumor suppressor genes and to overcome drug resistance also occurring in cancers addicted to actionable drivers. The discovery of non-oncogene addiction (NOA) uncovered novel therapeutically exploitable "Achilles' heels". NOA refers to genes/pathways not oncogenic per sé but essential for the tumor cell growth/survival while dispensable for normal cells. The clinical success of several classes of conventional and molecular targeted agents can be ascribed to their impact on both tumor cell-associated intrinsic as well as microenvironment-related extrinsic NOA. The integration of genetic, computational and pharmacological high-throughput approaches led to the identification of an expanded repertoire of synthetic lethality interactions implicating NOA targets. Only a few of them have been translated into the clinics as most NOA vulnerabilities are not easily druggable or appealing targets. Nonetheless, their identification has provided in-depth knowledge of tumor pathobiology and suggested novel therapeutic opportunities. Here, we summarize conceptual framework of intrinsic and extrinsic NOA providing exploitable vulnerabilities. Conventional and emerging methodological approaches used to disclose NOA dependencies are reported together with their limits. We illustrate NOA paradigmatic and peculiar examples and outline the functional/mechanistic aspects, potential druggability and translational interest. Finally, we comment on difficulties in exploiting the NOA-generated knowledge to develop novel therapeutic approaches to be translated into the clinics and to fully harness the potential of clinically available drugs.

Senescent Thyrocytes, Similarly to Thyroid Tumor Cells, Elicit M2-like Macrophage Polarization In Vivo.

Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.

Targeting Non-Oncogene Addiction: Focus on Thyroid Cancer.

Thyroid carcinoma (TC) is the most common malignancy of endocrine organs with an increasing incidence in industrialized countries. The majority of TC are characterized by a good prognosis, even though cases with aggressive forms not cured by standard therapies are also present. Moreover, target therapies have led to low rates of partial response and prompted the emergence of resistance, indicating that new therapies are needed. In this review, we summarize current literature about the non-oncogene addiction (NOA) concept, which indicates that cancer cells, at variance with normal cells, rely on the activity of genes, usually not mutated or aberrantly expressed, essential for coping with the transformed phenotype. We highlight the potential of non-oncogenes as a point of intervention for cancer therapy in general, and present evidence for new putative non-oncogenes that are essential for TC survival and that may constitute attractive new therapeutic targets.

COPZ1 depletion in thyroid tumor cells triggers type I IFN response and immunogenic cell death.

The coatomer protein complex zeta 1 (COPZ1) represents a non-oncogene addiction for thyroid cancer (TC); its depletion impairs the viability of thyroid tumor cells, leads to abortive autophagy, ER stress, UPR and apoptosis, and reduces tumor growth of TC xenograft models. In this study we investigated the molecular pathways activated by COPZ1 depletion and the paracrine effects on cellular microenvironment and immune response. By comprehensive and target approaches we demonstrated that COPZ1 depletion in TPC-1 and 8505C thyroid tumor cell lines activates type I IFN pathway and viral mimicry responses. The secretome from COPZ1-depleted cells was enriched for several inflammatory molecules and damage-associated molecular patterns (DAMPs). Moreover, we found that dendritic cells, exposed to these secretomes, expressed high levels of differentiation and maturation markers, and stimulated the proliferation of naïve T cells. Interestingly, T cells stimulated with COPZ1-depleted cells showed increased cytotoxic activity against parental tumor cells. Collectively, our findings support the notion that targeting COPZ1 may represent a promising therapeutic approach for TC, considering its specificity for cancer cells, the lack of effect on normal cells, and the capacity to prompt an anti-tumor immune response.

Mitosis perturbation by MASTL depletion impairs the viability of thyroid tumor cells.

Even if thyroid tumors are generally curable, a fraction will develop resistance to therapy and progress towards undifferentiated forms, whose treatment remains a demanding challenge. To identify potential novel targets for treatment of thyroid cancer, in a previous study using siRNA-mediated functional screening, we identified several genes that are essential for the growth of thyroid tumor, but not normal cells. Among the top-ranking hits, we found microtubule associated serine/threonine kinase-like (MASTL), which is known to play an essential role in mitosis regulation, and is also involved in the DNA damage response. Herein, we examine the effects of MASTL depletion on growth and viability of thyroid tumor cells. MASTL depletion impaired cell proliferation and increased the percentage of cells presenting nuclear anomalies, which are indicative of mitotic catastrophe. Furthermore, MASTL depletion was associated with enhanced DNA damage. All these effects eventually led to cell death, characterized by the presence of apoptotic markers. Moreover, MASTL depletion sensitized thyroid tumor cells to cisplatin. Our results demonstrate that MASTL represents vulnerability for thyroid tumor cells, which could be explored as a therapeutic target for thyroid cancer.

Senescent thyrocytes and thyroid tumor cells induce M2-like macrophage polarization of human monocytes via a PGE2-dependent mechanism.

BACKGROUND: Thyroid carcinoma includes several variants characterized by different biological and clinical features: from indolent microcarcinoma to undifferentiated and aggressive anaplastic carcinoma. Inflammation plays a critical role in thyroid tumors. Conditions predisposing to cancer, as well as oncogene activity, contribute to the construction of an inflammatory microenvironment that facilitates thyroid tumor progression. Moreover, oncogene-induced senescence, a mechanism tightly connected with inflammation, and able to restrain or promote cancer progression, is involved in thyroid cancer. The interactions between thyroid tumor cells and the microenvironment are not completely clarified. METHODS: We characterize in vitro the interplay between macrophages and senescent thyrocytes and tumor-derived cell lines, modeling early and late thyroid tumor stages, respectively. Purified peripheral blood-derived human monocytes were exposed to thyroid cell-derived conditioned medium (CM) and assessed for phenotype by flow cytometry. The factors secreted by thyroid cells and macrophages were identified by gene expression analysis and ELISA. The protumoral effect of macrophages was assessed by wound healing assay on K1 thyroid tumor cells. The expression of PTGS2 and M2 markers in thyroid tumors was investigated in publicly available datasets. RESULTS: Human monocytes exposed to CM from senescent thyrocytes and thyroid tumor cell lines undergo M2-like polarization, showing high CD206 and low MHC II markers, and upregulation of CCL17 secretion. The obtained M2-like macrophages displayed tumor-promoting activity. Among genes overexpressed in polarizing cells, we identified the prostaglandin-endoperoxide synthase enzyme (PTGS2/COX-2), which is involved in the production of prostaglandin E2 (PGE2). By using COX-2 inhibitors we demonstrated that the M2-like polarization ability of thyroid cells is related to the production of PGE2. Co-expression of PTGS2 and M2 markers is observed a significant fraction of human thyroid tumors. CONCLUSIONS: Our results demonstrate that both senescent thyrocytes and thyroid tumor cell lines trigger M2-like macrophage polarization that is related to PGE2 secretion. This suggests that the interaction with the microenvironment occurs at both early and late thyroid tumor stages, and favors tumor progression. The co-expression of PTGS2 gene and M2 markers in human thyroid carcinoma highlights the possibility to counteract tumor growth through COX-2 inhibition.

Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening.

The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3-1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies.

Molecular spectrum of BRAF, NRAS and KRAS gene mutations in plasma cell dyscrasias: implication for MEK-ERK pathway activation.

Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell (PC) malignancy. Whole-exome sequencing has identified therapeutically targetable mutations such as those in the mitogen-activated protein kinase (MAPK) pathway, which are the most prevalent MM mutations. We used deep sequencing to screen 167 representative patients with PC dyscrasias [132 with MM, 24 with primary PC leukemia (pPCL) and 11 with secondary PC leukemia (sPCL)] for mutations in BRAF, NRAS and KRAS, which were respectively found in 12%, 23.9% and 29.3% of cases. Overall, the MAPK pathway was affected in 57.5% of the patients (63.6% of those with sPCL, 59.8% of those with MM, and 41.7% of those with pPCL). The majority of BRAF variants were comparably expressed at transcript level. Additionally, gene expression profiling indicated the MAPK pathway is activated in mutated patients. Finally, we found that vemurafenib inhibition of BRAF activation in mutated U266 cells affected the expression of genes known to be associated with MM. Our data confirm and extend previous published evidence that MAPK pathway activation is recurrent in myeloma; the finding that it is mediated by BRAF mutations in a significant fraction of patients has potentially immediate clinical implications.

Oncogenic RAS-induced senescence in human primary thyrocytes: molecular effectors and inflammatory secretome involved.

Oncogene-induced senescence (OIS) is a robust and sustained antiproliferative response to oncogenic stress and constitutes an efficient barrier to tumour progression. We have recently proposed that OIS may be involved in the pathogenesis of thyroid carcinoma by restraining tumour progression as well as the transition of well differentiated to more aggressive variants. Here, an OIS inducible model was established and used for dissecting the molecular mechanisms and players regulating senescence in human primary thyrocytes. We show that oncogenic RAS induces senescence in thyrocytes as judged by changes in cell morphology, activation of p16INK4a and p53/p21CIP1 tumour suppressor pathways, senescence-associated ß-galactosidase (SA-ß-Gal) activity, and induction of proinflammatory components including IL-8 and its receptor CXCR2. Using RNA interference (RNAi) we demonstrate that p16INK4a is necessary for the onset of senescence in primary thyrocytes as its depletion rescues RAS-induced senescence. Furthermore, we found that IL-8/CXCR2 network reinforces the growth arrest triggered by oncogenic RAS, as its abrogation is enough to resume proliferation. Importantly, we observed that CXCR2 expression coexists with OIS markers in thyroid tumour samples, suggesting that CXCR2 contributes to senescence, thus limiting thyroid tumour progression.

Diagnostic utility of MS-MLPA in DNA methylation profiling of adenocarcinomas and neuroendocrine carcinomas of the colon-rectum.

Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) is a fast, new, inexpensive method that has rarely been exploited in DNA methylation profiling of colorectal cancers (CRCs). The aim of this study was to test the diagnostic utility of MS-MLPA to evaluate the methylation status of 34 genes in normal colonic mucosa samples and in a well-characterized series of 83 adenocarcinomas and 21 neuroendocrine carcinomas of colon-rectum. Two commercial MS-MLPA kits (SALSA MS-MLPA ME001-C1 Tumor suppressor-1 Kit and SALSA MS-MLPA ME002-B1 Tumor suppressor-2 Kit) were used to perform promoter methylation analysis on formalin-fixed and paraffin-embedded tissues. MS-MLPA analysis was validated by bisulfite pyrosequencing, bisulfite cycle sequencing, and methylation-specific PCR. MS-MLPA analysis identified a subset of 27 CRCs (26 % of cases) showing high levels of gene methylation involving a mean percentage of 34 % of the promoters examined. These tumors exhibited all the main clinicopathological and genetic features described for CRCs with CpG island Methylator Phenotype-High. High levels of methylation were observed with similar frequency in adenocarcinomas and in neuroendocrine carcinomas (25 % versus 29 %, respectively), but different methylation profiles were observed in the two tumor types. In both groups, tumors with microsatellite instability and widespread methylation represented a homogeneous clinicopathological entity. MS-MLPA assay is an easy and reliable system for epigenetic characterization of tumor tissues and leads to a rapid identification of CRCs with the highest levels of gene methylation. Aberrant gene methylation is a common abnormality in CRC initiation and may be observed in tumors with very different genetic and clinicopathological profiles.

S100A11 overexpression contributes to the malignant phenotype of papillary thyroid carcinoma.

CONTEXT: Papillary thyroid carcinoma (PTC) is the most frequent thyroid tumor and is responsible for the overall increase in thyroid cancer incidence. S100A11 (calgizzarin), a member of the S100 Ca(2+)-binding protein family, is involved in several different biological processes. S100A11 has been found up-regulated in PTC, both at the mRNA and protein levels. OBJECTIVE: Through a combination of expression analysis and functional in vitro and in vivo studies, we have attempted to gain insight into the relevance of S100A11 overexpression in PTC biology. DESIGN: The expression of the S100A11 gene in PTC was investigated in several gene expression data sets. The effect of S100A11 silencing on the hallmarks of the malignant phenotype of several PTC-derived cell lines was investigated. In NIH3T3 cells, the cooperation of S100A11 with the different PTC-specific oncogenes was assessed. RESULTS: We found that the S100A11 gene expression is frequently up-regulated in PTC, anaplastic thyroid carcinoma, but not in follicular thyroid carcinoma. S100A11 overexpression was also detected in PTC-derived cell lines, which were then used for functional studies. S100A11 silencing in PTC-derived cell lines did not affect cell proliferation, whereas it reduced the loss of contact inhibition, anchorage-independent growth, and resistance to anoikis. Cotransfection experiments in NIH3T3 cells showed that overexpression of the S100A11 gene was able to enhance the transforming capabilities of the different PTC-associated oncogenes by affecting the loss of contact inhibition, anchorage-independent growth, and in vivo tumor formation. CONCLUSION: Our data indicate that S100A11 overexpression exerts a protumoral functional role in PTC pathogenesis.

Are two better than one? A novel double-mutant KIT in GIST that responds to Imatinib.

Gastrointestinal stromal tumors carry in about 85% of the cases activating mutations in KIT gene. Generally only one KIT mutation is found in primary tumors and the majority of mutations affecting KIT exon 11 is sensitive to Imatinib. We report upon a GIST case harboring a double-mutant KIT gene at exon 11, which expresses a receptor bearing the known activating W557G mutation and a newly discovered missense Y578C alteration. The relative affinities for ATP and Imatinib of each single (W557G, Y578C) and double (W557G/Y578C) mutant KITs were predicted by in silico studies (computer-based molecular simulations), and compared with those obtained for known Imatinib sensitive and resistant KIT mutants. In parallel, biochemical analysis of the single and double KIT mutants expressed in mammalian cells was performed. Both the in-silico/in-vitro investigations showed constitutive activation and sensitivity to Imatinib of the yet mentioned Y578C mutation as well as of the double mutant, providing evidence that the concomitant presence of the W557G and Y578C mutations does not affect Imatinib response compare to the single mutations, in line with what observed in Imatinib treated patient.