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The insulin receptor isoform A (IR-A) plays an increasingly recognized role in fetal growth and tumor biology in response to circulating insulin and/or locally produced IGF2. This role seems not to be shared by the IR isoform B (IR-B). We aimed to dissect the specific impact of IR isoforms in modulating insulin signaling in triple negative breast cancer (TNBC) cells. We generated murine 4T1 TNBC cells deleted from the endogenous insulin receptor (INSR) gene and expressing comparable levels of either human IR-A or IR-B. We then measured IR isoform-specific in vitro and in vivo biological effects and transcriptome in response to insulin. Overall, the IR-A was more potent than the IR-B in mediating cell migration, invasion, and in vivo tumor growth. Transcriptome analysis showed that approximately 89% of insulin-stimulated transcripts depended solely on the expression of the specific isoform. Notably, in cells overexpressing IR-A, insulin strongly induced genes involved in tumor progression and immune evasion including chemokines and genes related to innate immunity. Conversely, in IR-B overexpressing cells, insulin predominantly induced the expression of genes primarily involved in the regulation of metabolic pathways and, to a lesser extent, tumor growth and angiogenesis.

The insulin receptor isoform A (IR-A), a dual receptor for insulin and IGF2, plays a role in breast cancer (BC) progression and metabolic reprogramming. Notably, discoidin domain receptor 1 (DDR1), a collagen receptor often dysregulated in cancer, is involved in a functional crosstalk and feed forward loop with both the IR-A and the insulin like growth factor receptor 1 (IGF1R). Here, we aimed at investigating whether DDR1 might affect BC cell metabolism by modulating the IGF1R and/or the IR. To this aim, we generated MCF7 BC cells engineered to stably overexpress either IGF2 (MCF7/IGF2) or the IR-A (MCF7/IR-A). In both cell models, we observed that DDR1 silencing induced a significant decrease of total ATP production, particularly affecting the rate of mitochondrial ATP production. We also observed the downregulation of key molecules implicated in both glycolysis and oxidative phosphorylation. These metabolic changes were not modulated by DDR1 binding to collagen and occurred in part in the absence of IR/IGF1R phosphorylation. DDR1 silencing was ineffective in MCF7 knocked out for DDR1. Taken together, these results indicate that DDR1, acting in part independently of IR/IGF1R stimulation, might work as a novel regulator of BC metabolism and should be considered as putative target for therapy in BC.

The development and progression of the great majority of breast cancers (BCs) are mainly dependent on the biological action elicited by estrogens through the classical estrogen receptor (ER), as well as the alternate receptor named G-protein–coupled estrogen receptor (GPER). In addition to estrogens, other hormones and growth factors, including the insulin and insulin-like growth factor system (IIGFs), play a role in BC. IIGFs cooperates with estrogen signaling to generate a multilevel cross-communication that ultimately facilitates the transition toward aggressive and life-threatening BC phenotypes. In this regard, the majority of BC deaths are correlated with the formation of metastatic lesions at distant sites. A thorough scrutiny of the biological and biochemical events orchestrating metastasis formation and dissemination has shown that virtually all cell types within the tumor microenvironment work closely with BC cells to seed cancerous units at distant sites. By establishing an intricate scheme of paracrine interactions that lead to the expression of genes involved in metastasis initiation, progression, and virulence, the cross-talk between BC cells and the surrounding microenvironmental components does dictate tumor fate and patients’ prognosis. Following (i) a description of the main microenvironmental events prompting BC metastases and (ii) a concise overview of estrogen and the IIGFs signaling and their major regulatory functions in BC, here we provide a comprehensive analysis of the most recent findings on the role of these transduction pathways toward metastatic dissemination. In particular, we focused our attention on the main microenvironmental targets of the estrogen-IIGFs interplay, and we recapitulated relevant molecular nodes that orientate shared biological responses fostering the metastatic program. On the basis of available studies, we propose that a functional cross-talk between estrogens and IIGFs, by affecting the BC microenvironment, may contribute to the metastatic process and may be regarded as a novel target for combination therapies aimed at preventing the metastatic evolution.

In the last decades increasing importance has been attributed to the Insulin/Insulin-like Growth Factor signaling (IIGFs) in cancer development, progression and resistance to therapy. In fact, IIGFs is often deregulated in cancer. In particular, the mitogenic insulin receptor isoform A (IR-A) and the insulin-like growth factor receptor (IGF-1R) are frequently overexpressed in cancer together with their cognate ligands IGF-1 and IGF-2. Recently, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis. Herein, we review recent findings indicating that DDR1 is as a novel modulator of IR and IGF-1R expression and function. DDR1 functionally interacts with IR and IGF-1R and enhances the biological actions of insulin, IGF-1 and IGF-2. Conversely, DDR1 is upregulated by IGF-1, IGF-2 and insulin through the PI3K/AKT/miR-199a-5p circuit. Furthermore, we discuss the role of the non-canonical estrogen receptor GPER1 in the DDR1-IIGFs crosstalk. These data suggest a wider role of DDR1 as a regulator of cell response to hormones, growth factors, and signals coming from the extracellular matrix.

The fetal isoform A of the insulin receptor (IR-A) is frequently overexpressed in a variety of malignancies including breast cancer. IR overexpression has a recognized role in cancer progression and resistance to anticancer therapies. In particular, IR-A has a peculiar mitogenic potential and is activated not only by insulin but also by IGF-2. Previously, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis. We now evaluated whether DDR1 is able to exert a role in breast cancer biology by functionally cross-talking with IR. In MCF-7 human breast cancer cells, IR and DDR1 co-immunoprecipitated and co-localized after insulin or IGF-2 stimulation. In a panel of breast cancer cells, DDR1 knockdown by specific siRNAs markedly inhibited IR downstream signaling as well as proliferation, migration and colony formation in response to insulin and IGF-2. These effects were accompanied by reduction of IR protein and mRNA expression, which involved both transcriptional and post-transcriptional effects. DDR1 overexpression elicited opposite effects. Bioinformatics analysis of public domain databases showed that IR and DDR1 co-expression significantly correlates with several clinically relevant histopathological and molecular features of human breast carcinomas. These findings demonstrate that, in human breast cancer cells, DDR1 regulates IR expression and ligand dependent biological actions. This novel functional crosstalk is likely clinically relevant and may become a new molecular target in breast cancer.

Previously published work has demonstrated that overexpression of the insulin receptor isoform A (IR-A) might play a role in cancer progression and metastasis. The IR has a predominant metabolic role in physiology, but the potential role of IR-A in cancer metabolic reprogramming is unknown. We aimed to characterize the metabolic impact of IR-A and its ligand insulin like growth factor 2 (IGF2) in human breast cancer (BC) cells. To establish autocrine IGF2 action, we generated human BC cells MCF7 overexpressing the human IGF2, while we focused on the metabolic effect of IR-A by stably infecting IGF1R-ablated MCF7 (MCF7IGF1R-ve) cells with a human IR-A cDNA. We then evaluated the expression of key metabolism related molecules and measured real-time extracellular acidification rates and oxygen consumption rates using the Seahorse technology. MCF7/IGF2 cells showed increased proliferation and invasion associated with aerobic glycolysis and mitochondrial biogenesis and activity. In MCF7IGF1R-ve/IR-A cells insulin and IGF2 stimulated similar metabolic changes and were equipotent in eliciting proliferative responses, while IGF2 more potently induced invasion. The combined treatment with the glycolysis inhibitor 2-deoxyglucose (2DG) and the mitochondrial inhibitor metformin blocked cell invasion and colony formation with additive effects. Overall, these results indicate that IGF2 and IR-A overexpression may contribute to BC metabolic reprogramming.

The insulin receptor (IR) mediates both metabolic and mitogenic effects especially when overexpressed or in clinical conditions with compensatory hyperinsulinemia, due to the metabolic pathway resistance, as obesity diabetes. In many cancers, IR is overexpressed preferentially as IR-A isoform, derived by alternative splicing of exon 11. The IR-A overexpression, and the increased IR-A:IR-B ratio, are mechanisms that promote the mitogenic response of cancer cells to insulin and IGF-2, which is produced locally by both epithelial and stromal cancer cells. In cancer IR-A, isoform predominance may occur for dysregulation at both mRNA transcription and post-transcription levels, including splicing factors, non-coding RNAs and protein degradation. The mechanisms that regulate IR isoform expression are complex and not fully understood. The IR isoform overexpression may play a role in cancer cell stemness, in tumor progression and in resistance to target therapies. From a clinical point of view, the IR-A overexpression in cancer may be a determinant factor for the resistance to IGF-1R target therapies for this issue. IR isoform expression in cancers may have the meaning of a predictive biomarker and co-targeting IGF-1R and IR-A may represent a new more efficacious treatment strategy.

Patients with thyroid cancers refractory to radioiodine (RAI) treatment show a limited response to various therapeutic options and a low survival rate. The recent use of multikinase inhibitors has also met limited success. An alternative approach relies on drugs that induce cell differentiation, as the ensuing increased expression of the cotransporter for sodium and iodine (NIS) may partially restore sensitivity to radioiodine. The inhibition of the ERK1/2 pathway has shown some efficacy in this context. Aggressive thyroid tumors overexpress the isoform-A of the insulin receptor (IR-A) and its ligand IGF-2; this IGF-2/IR-A loop is associated with de-differentiation and stem-like phenotype, resembling RAI-refractory tumors. Importantly, IR-A has been shown to be positively modulated by the non-integrin collagen receptor DDR1 in human breast cancer. Using undifferentiated human thyroid cancer cells, we now evaluated the effects of DDR1 on IGF-2/IR-A loop and on markers of cell differentiation and stemness. DDR1 silencing or downregulation caused significant reduction of IR-A and IGF-2 expression, and concomitant increased levels of differentiation markers (NIS, Tg, TSH, TPO). Conversely, markers of epithelial-to-mesenchymal transition (Vimentin, Snail-2, Zeb1, Zeb2 and N-Cadherin) and stemness (OCT-4, SOX-2, ABCG2 and Nanog) decreased. These effects were collagen independent. In contrast, overexpression of either DDR1 or its kinase-inactive variant K618A DDR1-induced changes suggestive of less differentiated and stem-like phenotype. Collagen stimulation was uneffective. In conclusion, in poorly differentiated thyroid cancer, DDR1 silencing or downregulation blocks the IGF-2/IR-A autocrine loop and induces cellular differentiation. These results may open novel therapeutic approaches for thyroid cancer.

Aims Hyperinsulinemia is a recognized risk factor for cancer and plays a major role for the increased cancer incidence in diabetic patients. Whether insulin analogs, and particularly long-acting analogs, worsen the pro-cancer effect of excess insulin is still controversial. Data synthesis In this paper we summarize the biological bases for the potential detrimental effect of long-acting analogs on cancer cells and review the in vitro and in vivo evidence on this issue. Because of their different molecular structure relative to native insulin, insulin analogs may activate the insulin receptor (IR) and the post receptor pathways differently. Most, but not all, in vitro evidence indicate that long-acting analogs may have a stronger mitogenic potency than insulin on cancer cells. Notably insulin glargine, the most studied long-acting analog, also has a higher affinity for the insulin-like growth factor (IGF)-1 receptor, a potent growth mediator. In vitro observations, however, may not reflect what occurs in vivo when analogs are metabolized to derivatives with a different mitogenic activity. Clinical studies, mostly retrospective and predominantly concerning glargine, provide contrasting results. The only perspective trial found no cancer increase in patients treated with glargine. All these studies, however, have severe weaknesses because of the insufficient evaluation of important factors such as dose administered, length of exposure, patient follow-up duration and site-specific cancer investigation. Moreover, whether cancer promotion is a long-acting analog class characteristic or a specific effect of a single agent is not clear. Conclusions In conclusion the carcinogenic risk of long-acting analogs, and specifically glargine, can be neither confirmed nor excluded. A personalized and shared decision, considering all the individual risk factors (metabolic and non-metabolic), is the suggestion for the clinician.

Thyroid cancer (TC) is the most common endocrine tumor. Although the majority of TCs show good prognoses, a minor proportion are aggressive and refractory to conventional therapies. So far, the molecular mechanisms underlying TC pathogenesis are incompletely understood. Evidence suggests that TC cells and their precursors are responsive to insulin and insulin-like growth factors (IGFs), and often overexpress receptors for insulin (IR) and IGF-1 (IGF-1R). IR exists in two isoforms, namely IR-A and IR-B. The first binds insulin and IGF-2, unlike IR-B, which only binds insulin. IR-A is preferentially expressed in prenatal life and contributes to development through IGF-2 action. Aggressive TC overexpresses IR-A, IGF-2, and IGF-1R. The over-activation of IR-A/IGF-2 loop in TC is associated with stem-like features and refractoriness to some targeted therapies. Importantly, both IR isoforms crosstalk with IGF-1R, giving rise to the formation of hybrids receptors (HR-A or HR-B). Other interactions have been demonstrated with other molecules such as the non-integrin collagen receptor, discoidin domain receptor 1 (DDR1), and the receptor for the hepatocyte growth factor (HGF), Met. These functional networks provide mechanisms for IR signaling diversification, which may also exert a role in TC stem cell biology, thereby contributing to TC initiation and progression. This review focuses on the molecular mechanisms by which deregulated IR isoforms and their crosstalk with other molecules and signaling pathways in TC cells and their precursors may contribute to thyroid carcinogenesis, progression, and resistance to conventional treatments. We also highlight how targeting these alterations starting from TC progenitors cells may represent new therapeutic strategies to improve the clinical management of advanced TCs.

Context: Cancer stem cells from several human malignancies, including poorly differentiated thyroid carcinoma and thyroid cancer cell lines, have been cultured in vitro as sphere-forming cells. These thyroid cancer stem cells were proven to be able to reproduce the original tumor in a xenograft orthotopic model. Objectives: The objective of the study was to characterize papillary thyroid carcinoma (PTC) spheres from well-differentiated thyroid cancer and normal thyroid (NT) spheres obtained from the contralateral thyroid tissue of the same patient. Design: Thyrospheres from PTCs and NTs were isolated. Main Outcome Measures: Gene expression analysis by real-time PCR, immunofluorescence studies, and fluorescence-activated cell sorter analysis in thyrospheres from PTCs and NTs have been evaluated. Conclusions: Compared with NT spheres, PTC spheres are larger, more irregular, and more clonogenic and have a higher rate of symmetric division. Moreover, PTC spheres express higher levels of stem cell markers and lower levels of thyroid-specific genes compared with NT spheres. Under appropriate conditions, NT spheres differentiated into thyrocytes, whereas PTC spheres did not, displaying a defect in the differentiation potential. Immunofluorescence experiments indicated that, in NT spheres, progenitor cells are mainly present in the sphere core, and the sphere periphery contains thyroid precursor cells already committed to differentiation. PTC spheres are not polarized like NT spheres. Unlike cells differentiated from NT spheres, TSH did not significantly stimulate cAMP production in cells differentiated from PTC spheres. A microarray analysis performed in paired samples (NT and PTC spheres from the same patient) indicated that NT and PTC spheres display a gene expression pattern typical of stem/progenitor cells; however, compared with NT spheres, PTC spheres display a unique gene expression pattern that might be involved in PTC progression.

Thyroid cancer (TC) is the most frequent endocrine tumor with a growing incidence worldwide. Besides the improvement of diagnosis, TC increasing incidence is probably due to environmental factors and lifestyle modifications. The actual diagnostic criteria for TC classification are based on fine needle biopsy (FNAB) and histological examination following thyroidectomy. Since in some cases it is not possible to make a proper diagnosis, classical approach needs to be supported by additional biomarkers. Recently, new emphasis has been given to the altered cellular metabolism of proliferating cancer cells which require high amount of glucose for energy production and macromolecules biosynthesis. Also TC displays alteration of energy metabolism orchestrated by oncogenes activation and tumor suppressors inactivation leading to abnormal proliferation. Furthermore, TC shows significant metabolic heterogeneity within the tumor microenvironment and metabolic coupling between cancer and stromal cells. In this review we focus on the current knowledge of metabolic alterations of TC and speculate that targeting TC metabolism may improve current therapeutic protocols for poorly differentiated TC. Future studies will further deepen the actual understandings of the metabolic phenotype of TC cells and will give the chance to provide novel prognostic biomarkers and therapeutic targets in tumors with a more aggressive behavior.

It is now widely accepted that insulin resistance and compensatory hyperinsulinemia are associated to increased cancer incidence and mortality. Moreover, cancer development and progression as well as cancer resistance to traditional anticancer therapies are often linked to a deregulation/overactivation of the insulin-like growth factor (IGF) axis, which involves the autocrine/paracrine production of IGFs (IGF-I and IGF-II) and overexpression of their cognate receptors [IGF-I receptor, IGF-insulin receptor (IR), and IR]. Recently, new drugs targeting various IGF axis components have been developed. However, these drugs have several limitations including the occurrence of insulin resistance and compensatory hyperinsulinemia, which, in turn, may affect cancer cell growth and survival. Therefore, new therapeutic approaches are needed. In this regard, the pleiotropic effects of peroxisome proliferator activated receptor (PPAR)-γ agonists may have promising applications in cancer prevention and therapy. Indeed, activation of PPAR-γ by thiazolidinediones (TZDs) or other agonists may inhibit cell growth and proliferation by lowering circulating insulin and affecting key pathways of the Insulin/IGF axis, such as PI3K/mTOR, MAPK, and GSK3-β/Wnt/β-catenin cascades, which regulate cancer cell survival, cell reprogramming, and differentiation. In light of these evidences, TZDs and other PPAR-γ agonists may be exploited as potential preventive and therapeutic agents in tumors addicted to the activation of IGF axis or occurring in hyperinsulinemic patients. Unfortunately, clinical trials using PPAR-γ agonists as antineoplastic agents have reached conflicting results, possibly because they have not selected tumors with overactivated insulin/IGF-I axis or occurring in hyperinsulinemic patients. In conclusion, the use of PPAR-γ agonists in combined therapies of IGF-driven malignancies looks promising but requires future developments.

The insulin receptor (IR) gene undergoes differential splicing that generates two IR isoforms, IR-A and IR-B. The physiological roles of IR isoforms are incompletely understood and appear to be determined by their different binding affinities for insulin-like growth factors (IGFs), particularly for IGF-2. Predominant roles of IR-A in prenatal growth and development and of IR-B in metabolic regulation are well established. However, emerging evidence indicates that the differential expression of IR isoforms may also help explain the diversification of insulin and IGF signaling and actions in various organs and tissues by involving not only different ligand-binding affinities but also different membrane partitioning and trafficking and possibly different abilities to interact with a variety of molecular partners. Of note, dysregulation of the IR-A/IR-B ratio is associated with insulin resistance, aging, and increased proliferative activity of normal and neoplastic tissues and appears to sustain detrimental effects. This review discusses novel information that has generated remarkable progress in our understanding of the physiology of IR isoforms and their role in disease. We also focus on novel IR ligands and modulators that should now be considered as an important strategy for better and safer treatment of diabetes and cancer and possibly other IR-related diseases.

The insulin/insulin-like growth factor system (IIGFs) plays a fundamental role in the regulation of prenatal and postnatal growth, metabolism and homeostasis. As a consequence, dysregulation of this axis is associated with growth disturbance, type 2 diabetes, chronic inflammation and tumor progression. A functional crosstalk between IIGFs and discoidin domain receptors (DDRs) has been recently discovered. DDRs are non-integrin collagen receptors that canonically undergo slow and long-lasting autophosphorylation after binding to fibrillar collagen. While both DDR1 and DDR2 functionally interact with IIGFs, the crosstalk with DDR1 is so far better characterized. Notably, the IIGFs-DDR1 crosstalk presents a feed-forward mechanism, which does not require collagen binding, thus identifying novel non-canonical action of DDR1. Further studies are needed to fully explore the role of this IIGFs-DDRs functional loop as potential target in the treatment of inflammatory and neoplastic disorders.

The Hepatocyte Growth Factor (HGF) and its receptor Met are physiological regulators of cell migration. HGF and Met have also been implicated in tumor progression and metastasis. We show here that the tyrosine kinase inhibitor STI571 has a stimulatory effect on HGF-induced migration and branching morphogenesis in thyroid cancer but not in primary or immortalized thyroid epithelial cells. These stimulatory effects of STI571 are observed at a concentration that is clinically relevant. The STI571-enhanced motile response can be correlated with an increase in the Met receptor tyrosine phosphorylation as well as ERK and Akt activation by HGF. Interestingly, one of the targets of STI571, namely the c-Abl tyrosine kinase, is activated by HGF and is recruited at the migrating edge of thyroid cancer cells. These data suggests that c-Abl and/or STI571-inhibited tyrosine kinases can negatively regulate the Met receptor to restrain the motile response in thyroid cancer cells.

Poorly differentiated thyroid carcinomas are refractory to common anticancer therapies, and novel inhibitors are being tested in these deadly malignancies. The epidermal growth factor receptor (EGFR) tyrosine kinase represents an attractive target for treatment because it is up-regulated in thyroid cancer and plays a role in cancer progression. However, EGFR inhibitors have provided poor results in thyroid carcinomas.We evaluated the possible mechanism underlying the resistance of thyroid cancer cells to EGFR inhibitors.We tested the effect of the EGFR tyrosine kinase inhibitor gefitinib in a panel of thyroid cancer cell lines.We found that in most of the cell lines, although gefitinib inhibited EGFR phosphorylation, it was poorly effective in reducing cell viability. gefitinib, however, was able to inhibit epidermal growth factor-induced cell migration and matrix invasion. In most thyroid cancer cell lines, gefitinib significantly inhibited Akt phosphorylation by inhibiting EGFR activation, but it had limited or no effect on ERK phosphorylation. The poor cell response to gefitinib was associated with genetic alterations, leading to constitutive activation of the ERK pathway, including BRAF(V600E) and HRAS(G12A/Q61R) mutations and RET/PTC1 rearrangement. When BRAF(V600E)-positive thyroid cancer cells were incubated with the specific BRAF inhibitor PLX4032, sensitivity to gefitinib was restored. Similar results were obtained with rat sarcoma and RET/papillary thyroid cancer inhibitors.These results indicate that thyroid cancer resistance to gefitinib is due to the constitutive activation of the mitogenic pathway by either signals downstream of EGFR or other tyrosine kinase receptors. This resistance can be overcome by the combined use of selective inhibitors.

Discoidin Domain Receptor 1 (DDR1) is a collagen receptor tyrosine-kinase that is implicated in tumor progression by contributing to epithelial mesenchymal transition (EMT) and by enhancing tumor cell migration and invasion. We previously demonstrated that, in breast cancer cells, DDR1 functionally cross-talks with IGF-I receptor (IGF-IR), by affecting IGF-IR expression and trafficking. In these cells, DDR1 was able to enhance the stimulatory effects of IGF-I on mitogenesis and invasion suggesting that the DDR1-IGF-IR cross-talk may play a role in cancer progression. Herein, we evaluated whether IGF-I may in turn affect DDR1 expression. Indeed, we demonstrated that in breast cancer cells (MCF-7 and MDA-MB-231), IGF-I was able to upregulate DDR1 protein expression in a time and dose dependent manner. In contrast, DDR1 mRNA levels showed non-significant changes after stimulation with IGF-I. Moreover, we found that DDR1 protein upregulation requires the activation of the PI3K/Akt pathway, while the Erk1/2, the p70/mTOR and the PKC pathways are not involved. Indeed, IGF-I induced DDR1 protein upregulation through a post-trascriptional mechanism involving miR-199a-5p suppression via Akt. We also found that autocrine IGF-II, induced by stable IGF-II cDNA transfection, is also able to elicit significant DDR1 upregulation in breast cancer cells. Similar effects were induced by IGF-II secreted by human cancer associated fibroblasts. Finally, we showed that miR-199a-5p transfection in breast cancer cells, decreased DDR1 expression and affected the response to IGF-I in terms of intracellular signaling through the PI3K/Akt and Erk1/2 pathways, and biological effects such as cell proliferation and migration. These results demonstrate that the protumoral effects of IGF-I include downregulation of miR-199a-5p and consequent upregulation of DDR1 protein. Both these molecules may represent important targets for breast cancers with overactivation of the IGF-IR axis. Citation Format: Chiara Palladino, Roberta Matà, Maria Luisa Nicolosi, Anna Rita Lo Presti, Roberta Malaguarnera, Marco Ragusa, Daniela Sciortino, Andrea Morrione, Marcello Maggiolini, Veronica Vella, Antonino Belfiore. In breast cancer cells IGF-I induces upregulation of DDR1 by suppressing miR-199a-5p via the PI3K/Akt pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4612.

Factors involved in the biology of normal and cancer stem/precursor cells from the thyroid are unknown. Thyroid cancer cells are responsive to insulin and IGF-I and IGF-II and often overexpress the insulin receptor (IR) and the IGF-I receptor (IGF-IR).We investigated the role of IR isoforms (IR-A and IR-B), IGF-IR, and their ligands in thyroid follicular cell precursors both normal and malignant.We established cultures of follicular cell precursors as thyrospheres from three papillary thyroid cancers and the corresponding nonaffected tissues. The expression of IR, IGF-IR, and their ligands was evaluated by quantitative RT-PCR and, in one case, also by Western blot. The effects of insulin and IGFs on thyrosphere growth and self-renewal were evaluated.Thyrospheres were characterized by the expression of stem cell markers and low/absent thyroid specific markers. Thyrospheres from normal tissue, but not from cancer tissue, could be induced to differentiate. Both IR isoforms, IGF-IR, IGF-I and IGF-II, were expressed at high levels in thyrospheres and markedly decreased in differentiating cells. IR-A was the predominant isoform in thyrospheres, especially from cancer, while IR-B was predominant in differentiating cells. Cancer thyrosphere growth was stimulated by insulin and IGFs.Our data suggest that IR isoforms and IGF-IR play a role in the biology of follicular thyroid precursors. Cell differentiation is associated with marked changes in the expression of these receptors and cognate ligands. These data may provide insight for future differentiation therapies in thyroid cancer.

ΔNp73 is a N-terminally truncated p53 family member with a dominant negative function, which is upregulated in cancer. PTEN is a lipid phosphatase, which is involved in the attenuation of tyrosine kinase signaling. PTEN expression is increased by p53, and its function is blunted in several malignancies. Because in most of the thyroid carcinomas, ΔNp73α is upregulated, whereas PTEN expression down regulated, we investigated whether ΔNp73α may influence PTEN expression in this cell model. We found that ΔNp73α overexpression in thyroid cancer cells reduces PTEN expression, whereas ΔNp73α down-regulation by siRNA increases PTEN expression. Real-time PCR indicated that overexpression of ΔNp73α is able to reduce PTEN mRNA levels. Moreover, chromatin immunoprecipitation (ChIP) and luciferase assays indicated that ΔNp73α binds to −1031-779 region of the PTEN promoter, which is a different site than that for p53, thereby inhibiting promoter activity. Interestingly, also the transcriptionally active p73 isoforms (TAp73α and TAp73β) bound to this DNA sequence and, at variance with ΔNp73α, stimulated PTEN promoter activity to an extent similar to that of p53. In accordance with its effect on PTEN protein levels, ΔNp73α increased phospho-Akt protein content and, as a consequence, Mdm2-mediated p53 degradation. This effect of ΔNp73α resulted in increased thyroid cancer cell proliferation and reduced apoptosis and was reverted by the PI3-kinase inhibitor LY294002, indicating the role of Akt pathway in this effect. Taken together, these results indicate a novel p73 regulated mechanism for PTEN expression in thyroid cancer cells, and that, also through this mechanism, ΔNp73α exerts its protumorigenic effect. © 2008 Wiley-Liss, Inc.

p53 family proteins include p53 tumor suppressor, p63, and p73. Despite the high similarity in structure and function with p53, p63, and p73 function in tumor suppression is still controversial. Here, we show that TAp73α, a transcriptionally active p73 isoform, is able to synergize p53 tumor suppressor function in thyroid cancer cells. Indeed, depletion of p73 by small interfering RNA in thyroid cancer cells resulted in a reduced transcriptional activity of p53. Ectopic coexpression of both p53 and TAp73α in thyroid cancer cells resulted in increased transcription and tumor suppressor function compared with p53 or TAp73α alone, as well as in increased p53 protein levels. The enhancing effect of TAp73α on p53 activity is Mdm2 dependent because it is prevented by Mdm2 depletion by small interfering RNA. At least two mechanisms may explain the interference of TAp73α with p53 function. First, in thyroid cancer cells, TAp73α inhibits the effect of p53 on Mdm2 induction by antagonizing p53 at the Mdm2 promoter level. Second, a TAp73α mutant (G264W), which is devoid of DNA binding capability, is still able to increase p53 protein levels by competing with p53 for Mdm2 protein binding. Taken together, these results indicate that in thyroid cancer cells, TAp73α is able to increase p53 protein level and function by interfering with Mdm2-mediated p53 degradation. These results may be useful for designing gene therapies aimed at restoring a normal p53 function in thyroid cancer cells. (Mol Cancer Res 2008;6(1):64–77)

The p73alpha protein is a functional homolog of the p53 tumor suppressor. Although the TP53 gene is frequently mutated in human cancers, the TP73 gene is rarely inactivated. We have found that p73alpha is highly expressed in a significant fraction of anaplastic thyroid cancer, whereas it is not detectable in normal thyroid epithelial cells or in papillary and follicular thyroid cancer cells. Interestingly, the tumor suppression function of p73alpha is actively restrained in anaplastic thyroid cancer cells. We have also found that c-Abl tyrosine kinase, an activator of p73, is excluded from the nucleus of p73alpha-positive thyroid cancer cells; whereas c-Abl undergoes nuclear-cytoplasmic shuttling in normal thyroid and p73-negative thyroid cancer cells. We constructed an AblNuk-FK506-binding protein (FKBP) fusion protein to enforce the nuclear accumulation of an inducible Abl kinase. Activation of this nuclear AblNuk-FKBP by dimerization with AP20187 in anaplastic thyroid cancer cells increased the levels of p73alpha and p21Cip1 and caused p73-dependent apoptosis. These results suggest subcellular segregation of c-Abl from p73 to be a strategy for disrupting the tumor suppression function of p73alpha.

The insulin receptor (IR) occurs in two isoforms (IR-A and IR-B) resulting from alternative splicing of exon 11 of the gene. The IR-A isoform is predominantly expressed in fetal tissues and malignant cells and binds IGF-II with high affinity. We previously observed that IRs are overexpressed in thyroid cancer cells; now we evaluated whether these cells preferentially express IR-A and produce IGF-II, which would activate a growth-promoting autocrine loop. The IR content ranged 6.0 –52.6 ng/100 g cell membrane protein in thyroid cancer primary cultures (n 8) and permanent cell lines (n 6) vs. 1.2–1.7 in normal thyroid cells (n 11 primary cultures; P < 0.0001). IR-A isoform relative abundance ranged from 36 –79% in cancer cells (with the highest values in undifferentiated cancers) vs. 27–39% in normal cells. Similar results were obtained in normal vs. cancer thyroid tissue specimens. IGF-II caused IR autophosphorylation with an ED50 of 1.5– 40.0 nM in cancer cells vs. more than 100 nM in normal cells; IGF-II affinity correlated with the relative abundance of IR-A (r 0.628; P < 0.0001). IGF-II was expressed in all cancer cells, highly expressed in anaplastic cells, and less expressed in normal cells. In conclusion, malignant thyrocytes, especially when poorly differentiated, produce IGF-II and overexpress IR, predominantly as IGF-II-sensitive isoform A . A growth-promoting autocrine loop is activated, therefore, and may affect thyroid cancer biology. (J Clin Endocrinol Metab 87: 245–254, 2002)