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Figure 1. Phase contrast microscope images of C1 and C2 organoids. Figure 2. Ingenuity Pathway Analysis of C2 secretome. Figure 3. Western blot analysis of C2 organoids after 4-IPP treatments. (PDF 282 kb)

Antibodies for immunohistochemistry. Table S2. Antibodies for Western blot. Table S3. Metabolic changes after 4-IPP or metformin treatments. (PDF 313 kb)

Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer.

Background: Gastrointestinal stromal tumours (GISTs) have activating KIT or PDGFRA gene mutations. Imatinib mesylate, which targets KIT and PDGFRA, is effective in treating GISTs, but 90% of GIST patients become imatinib-resistant as a result of acquiring secondary KIT mutations. Recent findings suggest that tumour growth can be driven by mutated self-renewing progenitors known as cancer stem cells (CSCs), which are believed to be present in all neoplastic proliferations and are thought to accumulate mutations. It is therefore possible that the acquisition of secondary KIT mutations during imatinib treatment may occur in putative GIST CSCs. Methods: Using flow cytometry, in vivo murine xenografts and molecular characterization, we tried to identify putative GIST CSCs by looking for the occurrence of common CSC markers such as KIT, CD133, CD90, CD44, and CD34 in 18 surgical samples obtained from nine untreated and nine imatinib-treated KIT-mutated GIST patients. Results: The results indicated the homogeneous and previously unreported expression of CD133 (18/18), CD90 (15/16), and CD44 (12/14), together with KIT (18/18) and CD34 (13/18). This profile is similar to that identified in bone marrow mesenchymal progenitors and does not seem to be significantly modified by imatinib as only marginal changes in KIT and CD133 expression (P ≤0.05, Mann-Whitney test) were found in the treated samples. Conclusions: These findings suggest that GISTs are a clonal expansion of quite primitive cells that strictly depend on KIT oncogenic addiction, and have no cancer/stem cell component that can be detected by means of the antigens used in this study. © 2011 International Clinical Cytometry Society

We have previously demonstrated that chordomas express activated platelet-derived growth factor receptor (PDGFRB) and that treatment with imatinib, which is capable of switching off the activation of various receptor tyrosine kinases (RTKs) including PDGFRB, benefits a number of patients. The aim of this study was to identify the possible presence of other activated RTKs and their downstream signaling effectors. Cryopreserved material from 22 naive sporadic chordomas was investigated for the presence of activated RTKs and their cognate ligands and downstream signaling effectors by means of human phospho-RTK antibody arrays, Western blotting, and molecular analysis; immunohistochemistry and fluorescence in situ hybridization were used to analyze the corresponding formalin-fixed and paraffin-embedded samples. We detected activated PDGFRB, FLT3, and colony stimulating factor 1 receptor (CSF1R) of the PDGFR family and highly phosphorylated EGFR, HER2/neu, and (to a lesser extent) HER4 of the EGFR family. The detection of PDGFRB/PDGFB confirmed our previous data. The presence of activated EGFR was paralleled by the finding of high levels of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) and PDGFB co-expression and PDGFRB co-immunoprecipitation. Of the downstream effectors, the PI3K/AKT and RAS/MAPK pathways were both activated, thus leading to the phosphorylation of mammalian target of rapamycin (mTOR) and 4E-BP1 among the regulators involved in translational control. Taken together, our results (i) provide a rationale for tailored treatments targeting upstream activated receptors, including the PDGFR and EGFR families; (ii) support the idea that a combination of upstream antagonists and mTOR inhibitors enhances the control of tumor growth; and (iii) indicate that the 4E-BP1/eIF4E pathway is a major regulator of protein synthesis in chordoma.