Therapy-induced senescence (TIS) is a potential outcome of anti-cancer treatments, characterized by a stable cell cycle arrest. However, it is now widely accepted that this process acts as a double-edged sword: in fact, senescent cells are active drivers of cancer relapse, aggressiveness and metastasis, through the release of pro-inflammatory factors and the ability to resume proliferation. Therefore, selectively targeting TIS cells, a strategy named one-two punch approach, is crucial to avoid their harmful effects. This may become particularly important for those aggressive tumors that currently lack effective therapeutic options, such as dedifferentiated thyroid tumors. To this purpose, identifying targetable characteristics of TIS cells is essential for the development of new senotherapeutics. TIS is often associated with variations of the autophagic flux, therefore, we investigated the interplay between autophagy and TIS in thyroid cancer cells, to explore a new potential target for senotherapy. We demonstrate that TIS thyroid cancer cells do not always exhibit a sufficient enlargement of the lysosomal compartment to maintain autophagy function. The deficiency in lysosomal biogenesis is driven by the inability of TFEB, the master regulator of this process, to properly enter and remain inside the nucleus. The disruption of the autophagic flux leads to the accumulation of SQSTM1/p62, which in turn activates the Nrf2 pathway. In contrast to cells with functional autophagy, Nrf2-activated cells display a higher tolerance to oxidative stress, making them resistant to the senolytic activity of lysosomal inhibitors.

ObjectiveIn the past years, the discovery of non-oncogene addiction (NOA) has expanded the cancer targets repertoire. In particular, novel strategies are aimed at counteracting the dependency of cancer cells on normal genes that are essential to sustain their stress phenotype. Of the NOA genes, discovered by our group, that are essential for human thyroid cancer cells, we extensively focused on COPZ1, a subunit of coatomer complex I. We previously reported that in in vitro human thyroid cancer models, COPZ1 depletion can induce IFN-I-mediated stimulatory effects, which culminates in immunogenic cell death. To evaluate in vivo the effects of COPZ1 depletion on immunity and inflammation, generation of syngeneic mouse models is needed. Toward this aim, in this work, we studied in vitro the dependency on COPZ1 in murine thyroid cancer cell lines. MethodsCell viability was assessed through crystal violet assay and a commercially available kit. qRT-PCR and western blot were used to evaluate the expression of genes and proteins of interest; commercially available kits were used for monitoring cytokine release and oxidative status. ResultsAnalogous to the human counterpart, COPZ1 silencing impaired cell proliferation and induced ER stress in four cell lines. The transcription and secretory IFN-I-related program was activated in two of the four cell lines. In the other two cell lines, we detected an increase in ROS production, lipid peroxidation, and Ca2+ levels. ConclusionActivation of inflammatory effects after COPZ1 silencing is cell line-dependent.

Non-oncogene addiction (NOA) indicates that tumor cell growth/survival requires the activity of genes/pathways not oncogenic per sé, and dispensable for normal cells. NOA genes provide a wide repertoire of novel therapeutic exploitable tumor vulnerabilities. A large body of evidence demonstrates the dependency of several tumors such as breast, prostate, ovary, thyroid, glioblastoma and LUAD, on the activity of COPZ1, a component of the heptameric COPI complex. Thus, COPZ1 is emerging as a potential novel therapeutic target for tumors of different origin. In different tumor models COPZ1 inhibition was found implicated in abortive autophagy, ER stress and activation of ferroptosis. In this review we summarize the different studies characterizing COPZ1 as a NOA gene in different tumor types, and discuss potential issues related to its targeting.

PURPOSE:The identification of novel molecular biomarkers may assist in the characterization of indeterminate thyroid nodules, which pose significant diagnostic challenges. Here, we aimed to explore the potential of proteomic analyses to support biomarker discovery in challenging thyroid lesions. METHODS:Linear Discriminant Analysis (LDA) was applied to Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) data from 44 thyroid neoplasms to select the most impactful molecular features for the classification of different tumor histologies, as well as for the distinction between NRAS-mutant (mNRAS) and NRAS-wild-type (wtNRAS) tumors. Relevant peaks were subsequently identified through nanoscale liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RESULTS:The LDA selected nine relevant molecular markers distinguishing noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs) from other tumor histologies (balanced accuracy = 73%), as well as 19 relevant markers able to identify mNRAS cases (balanced accuracy = 84%). Nine differentially expressed proteins were putatively identified: among them, ATP-dependent RNA helicase DDX42 showed a similar distribution between NIFTPs and papillary thyroid carcinomas (PTCs) / follicular variant PTCs (FVPTCs), while the distribution of the Histone H4 signal was similar between NIFTPs and follicular adenomas (FAs). In addition, Protein disulfide-isomerase A1 and Complement C4-B were overexpressed in wtNRAS compared to mNRAS cases, regardless of histology. CONCLUSION:The LDA-selected features enable to distinguish NIFTPs from morphologically similar lesions and to discriminate between mNRAS and wtNRAS cases. The identified markers might complement genetic analyses and provide insights into the distinct pathogenic drivers behind the development of mNRAS compared to wtNRAS lesions.

Therapy-induced senescence (TIS), is a potential outcome of anti-cancer treatments, characterized by a stable cell cycle arrest. However, it is now widely accepted that this process acts as a double-edged sword: in facts, senescent cells are active drivers of cancer relapse, aggressiveness and metastasis, through the release of various pro-inflammatory factors, and the ability to resume proliferation. Therefore, selectively targeting TIS cells, a strategy named one-two punch approach, is crucial to avoid their harmful effects. To this purpose, identifying targetable characteristics of TIS cells is essential for the development of new senotherapeutics. One of the peculiarities of senescent cells is the expansion of the lysosomal compartment. To assess whether lysosomes may be a valid target for new senotherapies, we employed as a TIS model different cell lines of anaplastic thyroid cancer, an aggressive and untreatable type of tumor, undergoing senescence upon either CDK4/6 inhibitor Palbociclib or ionizing radiation treatment. Senescence induction and lysosomal expansion were assessed by a multi-marker approach, comprising β-gal assay, γH2AX nuclear foci quantification, p21 and HMGA2 expression levels, and lysosomal staining. Our findings indicated that the response to TIS varied among cell lines regarding lysosomal content increase. Although the autophagic flux, that relies on lysosomes, was elevated across all cell lines, its functionality was line-specific, as evidenced by differential p62 protein turnover. Targeting autophagy with the lysosomal inhibitor Chloroquine exhibited a senolytic effect only on senescent cells with an expanded lysosomal compartment and functional autophagy. Conversely, the lysosome-sparing autophagy inhibitor LY294002 did not replicate these results, suggesting that the senolytic effect is ascribable to lysosomal rather than autophagy inhibition. To further explore the differential expansion of the lysosomal compartment, we analyzed the localization of TFEB, the master regulator of lysosomal biogenesis, following TIS induction, through immunofluorescence. Six days after treatment, TFEB maintained a nuclear localization exclusively in the cell line with functional autophagy. Interestingly, this was associated with significantly lower protein levels of Exportin-1, an export receptor responsible for TFEB nuclear extrusion. Our results indicate that lysosomes represent a promising target for developing new senotherapeutics, but TFEB’s ability to enter and persist at the nuclear level appears to be crucial in determining this sensitivity. Understanding the molecular differences underlying the varying functionality of the lysosomal biogenesis machinery, often defective in cancer, may pave the way for the future introduction of this one-two punch approach in the clinical setting. Beatrice Mazzoleni, Mara Mazzoni, Angela Greco. TFEB nuclear persistence predicts sensitivity to lysosomal inhibition in therapy-induced senescent thyroid cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 294.

Follicular-patterned thyroid neoplasms comprise a diverse group of lesions that pose significant challenges in terms of differential diagnosis based solely on morphologic and genetic features. Thus, the identification of easily testable biomarkers complementing microscopic and genetic analyses is a highly anticipated advancement that could improve diagnostic accuracy, particularly for noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs). These tumors exhibit considerable morphological and molecular heterogeneity, which may complicate their distinction from structurally similar neoplasms, especially when genetic analyses reveal shared genomic alterations (e.g., RAS mutations). Here, we integrated next-generation sequencing (NGS) with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to perform a proteogenomic analysis on 85 NIFTPs (n = 30 RAS-mutant [RAS-mut] and n = 55 RAS-wild type [RAS-wt]), with the aim to detect putative biomarkers of RAS-mut lesions. Through this combined approach, we identified four proteins that were significantly underexpressed in RAS-mut as compared to RAS-wt NIFTPs. These proteins could serve as readily accessible markers in morphologically borderline cases showing RAS mutations. Additionally, our findings may provide insights into the distinct pathogenic pathways through which RAS-mut and RAS-wt NIFTPs arise, highlighting the pivotal role of constitutive RAS-mitogen-activated protein kinase (MAPK) pathway activation in the development and progression of RAS-mut tumors.

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.

PDF file - 76K, Swiss GIST case material.

PDF file - 69K, Italian GIST case material.

PDF - 1320K, Contains Legends to Supplementary Figures; Supplementary Table 1: sequences for primers and siRNAs; Supplementary Figure 1: IC50 curves for chemoresistant cell lines; Supplementary Figure 2: Proliferation and dose-response curves for MT1G+ clones, as well as cell cycle analysis results; Supplementary Figure 3: MTs and labile zinc induction by extracellular zinc; Supplementary Figure 4: MTs immunofluorescence after chemotherapy or zinc treatments; Supplementary Figure 5: Western Blot of subcellular fractions; Supplementary Figure 6: Zinc transporter expression after chemotherapy; Supplementary Figure 7: Expression of p53 and NF-KB target genes after OXA treatment; Supplementary Figure 8: Cytotoxicity of zinc chloride.

Supplementary Figure Legends 1-6 from Antitumor and Anti-inflammatory Effects of Trabectedin on Human Myxoid Liposarcoma Cells

Supplementary Results and Supplementary Table S1.

Background: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer (TC). Several genomic and transcriptomic studies explored the molecular landscape of follicular cell-derived TCs, and BRAFV600E, RAS mutations, and gene fusions are well-established drivers. DICER1 mutations were described in specific sets of TC patients but represent a rare event in adult TC patients.Methods: Here, we report the molecular characterization of 30 retrospective follicular cell-derived thyroid tumors, comprising PTCs (90%) and poorly differentiated TCs (10%), collected at our Institute. We performed DNA whole-exome sequencing using patient-matched control for somatic mutation calling, and targeted RNA-seq for gene fusion detection. Transcriptional profiles established in the same cohort by microarray were investigated using three signaling-related gene signatures derived from The Cancer Genome Atlas (TCGA).Results: The occurrence of BRAFV600E (44%), RAS mutations (13%), and gene fusions (13%) was confirmed in our cohort. In addition, in two patients lacking known drivers, mutations of the DICER1 gene (p.D1709N and p.D1810V) were identified. DICER1 mutations occur in two adult patients with follicular-pattern lesions, and in one of them a second concurrent DICER1 mutation (p.R459*) is also observed. Additional putative drivers include ROS1 gene (p.P2130A mutation), identified in a patient with a rare solid-trabecular subtype of PTC. Transcriptomics indicates that DICER1 tumors are RAS-like, whereas the ROS1-mutated tumor displays a borderline RAS-/BRAF-like subtype. We also provide an overview of DICER1 and ROS1 mutations in thyroid lesions by investigating the COSMIC database.Conclusion: Even though small, our series recapitulates the genetic background of PTC. Furthermore, we identified DICER1 mutations, one of which is previously unreported in thyroid lesions. For these less common alterations and for patients with unknown drivers, we provide signaling information applying TCGA-derived classification.

Medullary thyroid carcinoma (MTC) is a rare but aggressive tumor. Although RET and RAS genes are recognized drivers in MTC, associated downstream signaling pathways are largely unknown. In this study, we report 17 sporadic MTCs, collected at our institution, comprising patient-matched primary and lymph node metastatic tumors investigated for mutational and transcriptional profiles. As we identified two uncommon RET deletions (D898_E901del and E632_L633del), we also performed a literature review and meta-analysis to assess the occurrence of unconventional alterations in MTC, focusing on next-generation sequencing studies. We found that new gene alterations are emerging, along with the known RET/RAS drivers, involving not only RET by multiple concurrent mutations or deletions but also other previously underestimated cancer-related genes, especially in sporadic MTCs. In our MTC gene profiles, we found transcriptome similarity between patient-matched tissues and expression of immune genes only by a few samples. Furthermore, we defined a gene signature able to stratify samples into two distinct signaling types, termed MEN2B-like and MEN2A-like. We provide an updated overview of the MTC mutational spectrum and describe how transcriptional profiles can be used to define distinct MTC signaling subtypes that appear to be shared by various gene drivers, including the unconventional ones.

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 β-galactosidase (SA-β-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.

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.

BRAFV600E is the most frequent oncogenic mutation identified in papillary thyroid cancer (PTC). In PTC patients who do not respond to standard treatment, BRAF inhibitors are currently tested as alternative strategies. However, as observed for other targeted therapies, patients eventually develop drug resistance. The mechanisms of BRAF inhibitors response are still poorly understood in a thyroid cancer (TC) context. In this study, we investigated in BRAFV600E mutated TC cell lines the effects of Vemurafenib and Dabrafenib, two BRAF inhibitors currently used in a clinical setting. We assessed cell proliferation, and the expression and activity of the thyroid function related transporter NIS following the treatment with BRAF inhibitors. In addition, we investigated the global gene expression by microarray, the relevant modulated biological processes by gene set enrichment analysis (GSEA), and TC specific gene signatures related to MAPK pathway activation, thyroid differentiation, and transcriptional profile associated with BRAFV600E or RAS mutation. We found that both inhibitors induce antiproliferative and redifferentiative effects on TC cells, as well as a rewiring of the MAPK pathway related to RAS signaling. Our results suggest a possible mechanism of drug response to the BRAF inhibitors Vemurafenib or Dabrafenib, supporting very recent findings in TC patients treated with targeted therapies.

Background Papillary thyroid cancer (PTC) is the most frequent endocrine tumor. Radioiodine (RAI) treatment is highly effective in these tumors, but up to 60% of metastatic cases become RAI-refractory. Scanty data are available on either the molecular pattern of radioiodine refractory papillary thyroid cancers (PTC) or the mechanisms responsible for RAI resistance. Methods We analyzed the molecular profile and gene/miRNA expression in primary PTCs, synchronous and RAI-refractory lymph node metastases (LNMs) in correlation to RAI avidity or refractoriness. We classified patients as RAI+/D+ (RAI uptake/disease persistence), RAI−/D+ (absent RAI uptake/disease persistence), and RAI+/D- (RAI uptake/disease remission), and analyzed the molecular and gene/miRNA profiles, and the expression of thyroid differentiation (TD) related genes. Results A different molecular profile according to the RAI class was observed: BRAF V600E cases were more frequent in RAI−/D+ ( P = 0.032), and fusion genes in RAI+/D+ cases. RAI+/D- patients were less frequently pTERT mutations positive, and more frequently wild type for the tested mutations/fusions. Expression profiles clearly distinguished PTC from normal thyroid. On the other hand, in refractory cases (RAI+/D+ and RAI−/D+) no distinctive PTC expression patterns were associated with either tissue type, or RAI uptake, but with the driving lesion and BRAF−/RAS-like subtype. Primary tumors and RAI-refractory LNMs with BRAF V600E mutation display transcriptome similarity suggesting that RAI minimally affects the expression profiles of RAI-refractory metastases. Molecular profiles associated with the expression of TPO , SLC26A4 and TD genes, that were found more downregulated in BRAF V600E than in gene fusions tumors. Conclusions The present data indicate a different molecular profile in RAI-avid and RAI-refractory metastatic PTCs. Moreover, BRAF V600E tumors displayed reduced differentiation and intrinsic RAI refractoriness, while PTCs with fusion oncogenes are RAI-avid but persistent, suggesting different oncogene-driven mechanisms leading to RAI refractoriness.

Thyroid carcinoma (TC) comprises several histotypes with different aggressiveness, from well (papillary carcinoma, PTC) to less differentiated forms (poorly differentiated and anaplastic thyroid carcinoma, PDTC and ATC, respectively). Previous reports have suggested a functional role for cancer-associated fibroblasts (CAFs) or senescent TC cells in the progression of PTC. In this study, we investigated the presence of CAFs and senescent cells in proprietary human TCs including PTC, PDTC, and ATC. Screening for the driving lesions BRAFV600E and N/H/KRAS mutations, and gene fusions was also performed to correlate results with tumor genotype. In samples with unidentified drivers, transcriptomic profiles were used to establish a BRAF- or RAS-like molecular subtype based on a gene signature derived from The Cancer Genome Atlas. By using immunohistochemistry, we found co-occurrence of stromal CAFs and senescent TC cells at the tumor invasive front, where deposition of collagen (COL1A1) and expression of lysyl oxidase (LOX) enzyme were also detected, in association with features of local invasion. Concurrent high expression of CAFs and of the senescent TC cells markers, COL1A1 and LOX was confirmed in different TC histotypes in proprietary and public gene sets derived from Gene Expression Omnibus (GEO) repository, and especially in BRAF mutated or BRAF-like tumors. In this study, we show that CAFs and senescent TC cells co-occur in various histotypes of BRAF-driven thyroid tumors and localize at the tumor invasive front.