Valentina Guarino

The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells

In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.

Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas.

To identify genes involved in the transformation of thyroid follicular cells, we explored, using DNA oligonucleotide microarrays, the transcriptional response of PC Cl3 rat thyroid epithelial cells to the ectopic expression of the RET/PTC oncogenes. We found that RET/PTC was able to induce the expression of CXCR4, the receptor for the chemokine CXCL12/SDF-1α/β. We observed that CXCR4 expression correlated with the transforming ability of the oncoprotein and depended on the integrity of the RET/PTC–RAS/ERK signaling pathway. We found that CXCR4 was expressed in RET/PTC-positive human thyroid cancer cell lines, but not in normal thyroid cells. Furthermore, we found CXCR4 expression in human thyroid carcinomas, but not in normal thyroid samples by immunohistochemistry. Since CXCR4 has been recently implicated in tumor proliferation, motility and invasiveness, we asked whether treatment with SDF-1α was able to induce a biological response in thyroid cells. We observed that SDF-1α induced S-phase entry and survival of thyroid cells. Invasion through a reconstituted extracellular matrix was also supported by SDF-1α and inhibited by a blocking antibody to CXCR4. Taken together, these results suggest that human thyroid cancers bearing RET/PTC rearrangements may use the CXCR4/SDF-1α receptor–ligand pathway to proliferate, survive and migrate.

Thyroid cancer and inflammation

Some cancer types are strongly associated with chronic inflammatory or infectious diseases whereas others are not, but an inflammatory component is present in most human neoplastic lesions. This review focuses on various aspects of thyroid cancer and inflammation. The incidence of thyroid cancer, in particular of well-differentiated papillary thyroid carcinomas (PTCs), is increased in autoimmune thyroid diseases such as Hashimotos thyroiditis. Thyroid cancer often has an inflammatory cell infiltrate, which includes lymphocytes, macrophages, dendritic cells and mast cells, whose role in thyroid cancer is still not completely understood. However, most experimental evidence suggests these cells exert a protumorigenic function. Moreover, oncoproteins typically expressed in human PTCs, such as RET/PTC, RAS, and BRAF, trigger a proinflammatory programme in thyreocytes. These data suggest that inflammatory molecules are promising targets for thyroid cancer therapy.

Biological Role and Potential Therapeutic Targeting of the Chemokine Receptor CXCR4 in Undifferentiated Thyroid Cancer

Anaplastic thyroid carcinoma (ATC) is a rare thyroid cancer type with an extremely poor prognosis. Despite appropriate treatment, which includes surgery, radiotherapy, and chemotherapy, this cancer is invariably fatal. CXCR4 is the receptor for the stromal cell-derived factor-1 (SDF-1)/CXCL12 chemokine and it is expressed in a variety of solid tumors, including papillary thyroid carcinoma. Here, we show that ATC cell lines overexpress CXCR4, both at the level of mRNA and protein. Furthermore, we found that CXCR4 was overexpressed in ATC clinical samples, with respect to normal thyroid tissues by real-time PCR and immunohistochemistry. Treatment of ATC cells with SDF-1 induced proliferation and increase in phosphorylation of extracellular signal-regulated kinases and protein kinase B/AKT. These effects were blocked by the specific CXCR4 antagonist AMD3100 and by CXCR4 RNA interference. Moreover, AMD3100 effectively reduced tumor growth in nude mice inoculated with different ATC cells. Thus, we suggest that CXCR4 targeting is a novel potential strategy in the treatment of human ATC.

Activation of TYRO3/AXL Tyrosine Kinase Receptors in Thyroid Cancer

Thyroid cancer is the most common endocrine cancer, but its key oncogenic drivers remain undefined. In this study we identified the TYRO3 and AXL receptor tyrosine kinases as transcriptional targets of the chemokine CXCL12/SDF-1 in CXCR4-expressing thyroid cancer cells. Both receptors were constitutively expressed in thyroid cancer cell lines but not normal thyroid cells. AXL displayed high levels of tyrosine phosphorylation in most cancer cell lines due to constitutive expression of its ligand GAS6. In human thyroid carcinoma specimens, but not in normal thyroid tissues, AXL and GAS6 were often coexpressed. In cell lines expressing both receptors and ligand, blocking each receptor or ligand dramatically affected cell viability and decreased resistance to apoptotic stimuli. Stimulation of GAS6-negative cancer cells with GAS6 increased their proliferation and survival. Similarly, siRNA-mediated silencing of AXL inhibited cancer cell viability, invasiveness, and growth of tumor xenografts in nude mice. Our findings suggest that a TYRO3/AXL-GAS6 autocrine circuit sustains the malignant features of thyroid cancer cells and that targeting the circuit could offer a novel therapeutic approach in this cancer.

Overexpression of the Cytokine Osteopontin Identifies Aggressive Laryngeal Squamous Cell Carcinomas and Enhances Carcinoma Cell Proliferation and Invasiveness

Purpose: Osteopontin is a secreted cytokine that binds to the cell surface CD44v6 receptor. We studied osteopontin and CD44v6 expression in laryngeal squamous cell carcinomas and correlated osteopontin expression levels with clinicopathologic tumor features. Experimental Design: We used immunohistochemistry, immunoblotting, and reverse transcription-PCR to study osteopontin expression in 58 laryngeal squamous cell carcinomas. Cultured squamous carcinoma cells were treated with exogenous osteopontin or with RNA interference to knockdown osteopontin expression. Results: Osteopontin expression was higher in all the invasive carcinomas than in patient-matched normal mucosa. Its expression levels were significantly correlated with tumor stage and grade and with the presence of lymph node and distant metastases. Osteopontin positivity was negatively correlated with overall survival (P = 0.03). Osteopontin expression was paralleled by intense cell surface reactivity for CD44v6. Treatment of squamous carcinoma cells with recombinant osteopontin sharply increased proliferation and Matrigel invasion in comparison with the untreated cells parallel to activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase signaling cascade. Osteopontin knockdown by RNA interference, anti-CD44 antibodies, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition prevented these effects. Conclusions: These results identify osteopontin as a marker and a potential therapeutic target in cases of aggressive laryngeal squamous cell carcinomas.

Autocrine stimulation by osteopontin plays a pivotal role in the expression of the mitogenic and invasive phenotype of RET/PTC-transformed thyroid cells

Papillary thyroid carcinomas are characterized by rearrangements of the RET receptor tyrosine kinase generating RET/PTC oncogenes. Here we show that osteopontin (OPN), a secreted glycoprotein, is a major RET/PTC-induced transcriptional target in PC Cl 3 thyroid follicular cells. OPN upregulation depended on the integrity of the RET/PTC kinase and tyrosines Y1015 and Y1062, two major RET/PTC autophosphorylation sites. RET/PTC also induced a strong overexpression of CD44, a cell surface signalling receptor for OPN. Upregulation of CD44 was dependent on RET/PTC Y1062, as well. Constitutive OPN overexpression or treatment with exogenous recombinant OPN sharply increased proliferation, Matrigel invasion and spreading in collagen gels of RET/PTC-transformed PC Cl 3 cells. These effects were impaired by the treatment of PC Cl 3-RET/PTC cells with OPN- and CD44-locking antibodies. Thus, RET/PTC signalling triggers an autocrine loop involving OPN and CD44 that sustains proliferation and invasion of transfomed PC Cl 3 thyrocytes.

OPN/CD44v6 overexpression in laryngeal dysplasia and correlation with clinical outcome

Laryngeal dysplasia is a common clinical concern. Despite major advancements, a significant number of patients with this condition progress to invasive squamous cell carcinoma. Osteopontin (OPN) is a secreted glycoprotein, whose expression is markedly elevated in several types of cancers. We explored OPN as a candidate biomarker for laryngeal dysplasia. To this aim, we examined OPN expression in 82 cases of dysplasia and in hyperplastic and normal tissue samples. OPN expression was elevated in all severe dysplasia samples, but not hyperplastic samples, with respect to matched normal mucosa. OPN expression levels correlated positively with degree of dysplasia (P=0.0094) and negatively with disease-free survival (P<0.0001). OPN expression was paralleled by cell surface reactivity for CD44v6, an OPN functional receptor. CD44v6 expression correlated negatively with disease-free survival, as well (P=0.0007). Taken as a whole, our finding identify OPN and CD44v6 as predictive markers of recurrence or aggressiveness in laryngeal intraepithelial neoplasia, and overall, point out an important signalling complex in the evolution of laryngeal dysplasia.

RAI(ShcC/N-Shc)-dependent recruitment of GAB1 to RET oncoproteins potentiates PI3-K signalling in thyroid tumors

RAI, also named ShcC/N-Shc, one of the members of the Shc proteins family, is a substrate of the RET receptor tyrosine kinase. Here, we show that RAI forms a protein complex with both RET/MEN2A and RET/PTC oncoproteins. By coimmunoprecipitation, we found that RAI associates with the Grb2-associated binder1 (GAB1) adapter. This association is constitutive, but, in the presence of RET oncoproteins, both RAI and GAB1 are tyrosine-phosphorylated, and the stoichiometry of this interaction remarkably increases. Consequently, the p85 regulatory subunit of phosphatidylinositol-3 kinase (PI-3K) is recruited to the complex, and its downstream effector Akt is activated. We show that human thyroid cancer cell lines derived from papillary or medullary thyroid carcinoma (PTC or MTC) carrying, respectively, RET/PTC and RET/MEN2A oncoproteins express RAI proteins. We also show that human PTC samples express higher levels of RAI, when compared to normal thyroid tissue. In thyroid cells expressing RET/PTC1, ectopic expression of RAI protects cells from apoptosis; on the other hand, the silencing of endogenous RAI by small inhibitory duplex RNAs in a PTC cell line that expresses endogenous RET/PTC1, increases the rate of spontaneous apoptosis. These data suggest that RAI is a critical substrate for RET oncoproteins in thyroid carcinomas.

Erratum: The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells (American Society for Clinical Investigation (2005) 115:4 (1068-1081) DOI: 10.1172/JCI22758)

Original citation: J Clin Invest. 2016;126(3):1067–1078. doi:10.1172/JCI82592. Citation for this erratum: J Clin Invest. 2016;126(4):1603. doi:10.1172/JCI87342. The scatter graph in Figure 4G was incorrect in the print version and the original online version of this article; a correct version of the latter has since been published. The correct figure panel is below. The JCI regrets the error. Erratum