Insulin Receptor Isoforms Differently Regulate Cell Proliferation and Apoptosis in the Ligand-Occupied and Unoccupied State

The insulin receptor (IR) presents two isoforms (IR-A and IR-B) that differ for the α-subunit C-terminal. Both isoforms are expressed in all human cells albeit in different proportions, yet their functional properties-when bound or unbound to insulin-are not well characterized. From a cell model deprived of the Insulin-like Growth Factor 1 Receptor (IGF1-R) we therefore generated cells exhibiting no IR (R-shIR cells), or only human IR-A (R-shIR-A), or exclusively human IR-B (R-shIR-B) and we studied the specific effect of the two isoforms on cell proliferation and cell apoptosis. In the absence of insulin both IR-A and IR-B similarly inhibited proliferation but IR-B was 2–3 fold more effective than IR-A in reducing resistance to etoposide-induced DNA damage. In the presence of insulin, IR-A and IR-B promoted proliferation with the former significantly more effective than the latter at increasing insulin concentrations. Moreover, only insulin-bound IR-A, but not IR-B, protected cells from etoposide-induced cytotoxicity. In conclusion, IR isoforms have different effects on cell proliferation and survival. When unoccupied, IR-A, which is predominantly expressed in undifferentiated and neoplastic cells, is less effective than IR-B in protecting cells from DNA damage. In the presence of insulin, particularly when present at high levels, IR-A provides a selective growth advantage.

SUN-131 The Roles of Two Insulin Receptor Isoforms in Triple Negative Breast Cancer Growth

Systemic hyperinsulinemia is believed to be an important factor in the progression of a number of cancers, including breast cancer by activating the insulin receptor (IR) signaling cascade in the tumor cells. The IR is expressed in two isoforms, IR-A and IR-B. IR-B is the full-length isoform, while IR-A is lacking 12 amino acids in the α-subunit due to exon 11 alternative splicing. IR-A is predominantly expressed in cancer tissues, while IR-B is mostly expressed in metabolic tissues. The IR and closely related insulin-like growth factor 1 receptor (IGF-1R) are expressed in different ratios in cancer cells. Compared with estrogen receptor positive breast cancers, triple negative breast cancers (TNBC) frequently have higher ratios of IR to IGF-1R. Hyperinsulinemia is associated with increased prevalence of TNBC in pre-menopausal women. Although new targeted therapies are emerging, among breast cancer subtypes TNBC continues to carry the worst prognosis and therefore developing a greater understanding of the links between IR signaling and TNBC progression is critical. The aim of this study is to understand the role of IR-A and IR-B on proliferation, metastasis and metabolism in breast cancer cells. We stably overexpressed human IR-A (IR-A OE) and IR-B (IR-B OE) in TNBC MDA-MB-231 (231) and murine c-myc/vegf overexpressing Mvt1 cells with lentiviral transduction using pLVX-IRES-puro HIV-1-based expression vectors with cDNA encoding the human IR-A,IR-B and control cDNA sequences. Native murine IR was silenced using lentiviral transduction of shRNA in the Mvt1 cells. Overexpression of IR was confirmed at a protein level by western blot, and RNA isoform expression was confirmed using real time PCR. Cell proliferation assays were performed in DMEM/10% FBS and revealed that MDA-MB-231 cells with IR-A OE cells had 15% higher proliferation rates than 231 IR-B OE cells. We then examined the IR signaling pathways by western blot in DMEM/10% FBS. No differences in phosphorylated or total ERK1/2 were observed between control, 231 IR-A OE and 231 IR-B OE cells. 231 IR-A OE cells were found to have 15-fold greater Akt phosphorylation (Ser473) than 231 control cells (p=0.0008) and 4 fold higher pAkt(Ser473) compared with 231 IR-B OE cells (p=0.0016). Further, we found that 231 IR-A OE cells had approximately 2 fold greater expression of c-myc protein compared with both 231 control (p=0.047) and 231 IR-B OE cells (p=0.026). No differences in c-myc expression were observed between 231 IRB OE and 231 control cells. In our previous studies we found that insulin stimulates c-myc expression and silencing the IR reduces c-myc expression in cancer cells. Our current studies show that IR-A, rather than IR-B is the insulin receptor isoform that regulates c-myc expression in human TNBC. Reference: (1) Belfiore et al., Endocr Relat Cancer. 2011; 18(4):R125-R147. (2) Ferguson et al., Breast Cancer Res. 2012; 14(1): R8.

Insulin-like growth factor-II in adipocyte regulation: depot-specific actions suggest a potential role limiting excess visceral adiposity

The IGF system has an important role in growth and development. IGF-II is a recognized fetal growth promoter. However, its physiological postnatal role remains uncertain, although it is maintained in the circulation at a substantially high level throughout life. IGF-II has been strongly linked to obesity in genetic studies, and more recent evidence suggests a metabolic role. We examined fat depot differences in IGF-II’s action on differentiation and metabolism. We speculate a specific effect on visceral adipocytes in relation to the differential distribution of insulin receptors between visceral and subcutaneous fat depots. We used a previously established adipocyte, cell culture system of matched pairs of visceral and subcutaneous fat biopsies from 20 normal weight children undergoing routine surgery for nonmalignant, nonseptic conditions. Preadipocytes were differentiated for 14 days in the presence or absence of IGF-II. Oil Red O staining, Western blotting, and reverse transcription polymerase chain reaction techniques were employed to assess levels of adipogenesis markers and levels of the insulin receptor and insulin receptor isoforms. Our data indicate that IGF-II promotes preadipocyte differentiation in subcutaneous preadipocytes but showed a protective, opposing effect restricting visceral preadipocyte differentiation, confirmed by reductions in the differentiation markers peroxisome proliferator-activated receptor gamma and adiponectin and in triglyceride staining. Additionally, IGF-II reduced mRNA expression of the insulin receptor in adipocytes and downregulated insulin receptor isoform A and glucose transporter 4 abundance and corresponding glucose uptake in visceral adipocytes. In conclusion, IGF-II is a regulator of preadipocyte differentiation and metabolism by acting as a differential modulator of fat accumulation favoring less visceral fat deposition in children.

The Emerging Role of Insulin Receptor Isoforms in Thyroid Cancer: Clinical Implications and New Perspectives

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.

Insulin Receptor Isoforms in Cancer

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.