Expression Of Insulin Receptor Isoforms Research Articles

Abstract B066: Elucidating the role of insulin receptor isoform expression in metastatic pancreatic ductal adenocarcinoma

Abstract Introduction: While type 2 diabetes mellitus (T2DM) has been shown to be both a risk factor and consequence of pancreatic ductal adenocarcinoma (PDAC), the bidirectional relationship between T2DM and PDAC remains to be fully understood. Due to spatial proximity of insulin-producing beta cells, PDAC tumor cells are presumably exposed to a highly concentrated insulin microenvironment, which may contribute to the dysregulation of the insulin receptor gene INSR. There are two isoforms of insulin receptor: IR-A and IR-B. IR-A activity has been associated with oncogenic function and is upregulated in several cancer types. This leads to questions regarding the role of INSR and more specifically, how the ratio of the two isoforms may potentiate tumor aggressiveness in PDAC. Results: Using data from whole-genome and RNA sequencing of metastatic PDAC (mPDAC) from both the PanGen (n=70; NCT01855477) and Personalized Oncogenomics (n=22; NCT02155621) trials, we identified somatic copy loss of INSR in 39/92 (42%) patient tumors, with the majority of such cases (35/39; 90%) showing heterozygous copy loss. There was no statistical association between INSR copy status and PDAC transcriptomic subtypes. INSR expression was lower in basal-like versus classical subtype tumors (p=1.1e-4), and INSR expression was further attenuated in basal-like tumors with heterozygous copy loss of INSR (p=0.0041). The ratio of IR-A:IR-B expression was heterogenous across samples, and heightened IR-A:IR-B ratio was significantly (p<0.05) associated with expression of genes linked to the PI3K-Akt signaling axis (CCDC88A, THEM4) and glucose metabolism (HK1, G6PC, PKLR) in basal-like tumors. Interestingly,HK1 and IGF1R were significantly upregulated, independent of IR-A:IR-B ratio, among basal-like (p 9.1e-9 and p=1.4e-5, respectively) compared to classical subtype tumors. Conclusion: These data indicate that heightened IR-A:IR-B ratio is associated with the expression of key pathways converging on PI3K signalling and glucose metabolism in mPDAC, while highlighting that several IR-A:IR-B-associated gene correlations are unique to basal-like tumors. Such findings warrant further investigation of relative INSR isoform expression in mPDAC and pre-clinical models to elucidate the role of the oncogenic isoform, IR-A, in driving tumor aggressiveness in basal-like subtype tumors. Citation Format: Lan V. Tao, James T. Topham, Joanna M. Karasinska, Erica S. Tsang, Andrew Metcalfe, Hassan Ali, Dawn Ashforth, Rachel Goodwin, Patricia A. Tang, Oliver F. Bathe, Janessa Laskin, Marco Marra, Daniel J. Renouf, David F. Schaeffer. Elucidating the role of insulin receptor isoform expression in metastatic pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B066.

Hyperinsulinemia induces early and dyssynchronous puberty in lean female mice.

Girls with obesity are at increased risk of early puberty. Obesity is associated with insulin resistance and hyperinsulinemia. We hypothesized that insulin plays a physiological role in pubertal transition, and super-imposed hyperinsulinemia due to childhood obesity promotes early initiation of puberty in girls. To isolate the effect of hyperinsulinemia from adiposity, we compared pre-pubertal and pubertal states in hyperinsulinemic, lean muscle (M)-insulin-like growth factor 1 receptor (IGF-1R)-lysine (K)-arginine (R) (MKR) mice to normoinsulinemic WT, with puberty onset defined by vaginal opening (VO). Our results show MKR had greater insulin resistance and higher insulin levels (P < 0.05) than WT despite lower body weight (P < 0.0001) and similar IGF-1 levels (P = NS). Serum luteinizing hormone (LH) levels were higher in hyperinsulinemic MKR (P = 0.005), and insulin stimulation induced an increase in LH levels in WT. VO was earlier in hyperinsulinemic MKR vs WT (P < 0.0001). When compared on the day of VO, kisspeptin expression was higher in hyperinsulinemic MKR vs WT (P < 0.05), and gonadotropin-releasing hormone and insulin receptor isoform expression was similar (P = NS). Despite accelerated VO, MKR had delayed, disordered ovarian follicle and mammary gland development. In conclusion, we found that hyperinsulinemia alone without adiposity triggers earlier puberty. In our study, hyperinsulinemia also promoted dyssynchrony between pubertal initiation and progression, urging future studies in girls with obesity to assess alterations in transition to adulthood.

Abstract P2-03-02: Detection of insulin receptor isoforms by mass spectrometry in breast cancer

Abstract Breast cancer cells express Type 1 Insulin-like Growth Factor Receptor (IGF1R) and Insulin Receptor (IR). These receptors have a role in breast cancer biology and mediate metabolism, proliferation, survival, and motility. IR gene undergoes differential splicing that generates two IR isoforms, IRA (exclusion of exon 11) and IRB. High levels of IRA expression in prenatal growth and development are observed, whereas IRB expression is more highly expressed in adult insulin responsive tissues. IRA overexpression is the predominant receptor isoform expressed in breast cancer specimens obtained from patients with endocrine resistant disease. To measure levels of IRA and IRB mRNA expression, we utilized IR isoform specific primers in quantitative reverse transcription PCR (qRT-PCR). The validation of IRA and IRB primers was determined via using IRA and IRB overexpressing clones of MCF7 cells. IRA and IRB mRNA expression was also studied in human adipose and liver tissues that majorly express the adult IRB isoform. Over 40 breast cancer cell lines and 20 ER+ patient tumor samples were studied to determine IRA and IRB mRNA expression. Total IR, IGF1R, IRS1, and IRS2 were also analyzed in cell lines and patient samples. Across the ATCC breast cancer cell lines and ER+ patient tumors, heterogeneity was found among all targeted genes. Using MCF-7 cells as control, we found one of the breast cancer cell lines, Du4475, has very high IRA mRNA expression level (200-fold of that in MCF-7). Using mass spectrometry technology, we quantified levels of IR isoform expression in Du4475 cells. Briefly, IR immunoprecipitates were subjected to SDS-PAGE gels and the IRA protein gel band was submitted for mass spectrometry. Since IRA does not contain exon 11, the identified unique peptide consistent with its expression is TFEDYLHNVVFVPRPS. After this IRA specific peptide was determined, the heavy-labeled (C13- and N15-containing) IRA peptide was used as an internal injection control to quantify the IRA level. Quantitative results of IRA peptode sequence were proportional to the mRNA levels previously detected in Du4475 cell lysate. Using MCF-7 IRB overexpression cell line MCF7L-IRB-C5, we identified two IRB peptides, KTSSGTGAEDPRPSR and TSSGTGAEDPRPSR at exon 11 region by mass spectrometry. Cell membrane and total cell lysates could also be used to detect IR protein isoforms by mass spectrometry analysis. Further measurement of IRA and IRB level on other breast cancer cell lines is ongoing. In summary, we are the first to use mass spectrometry to validate protein expression of IR isoforms at levels that correlate with mRNA expression. IRA mRNA and protein expression levels in breast cancer cell lines may serve as biomarkers to screen patients for targeted therapy. Citation Format: Xihong Zhang, LeeAnn Higgins, Todd Markowski, Tzu-Yi Yang, Jacob Wragge, Kevin Murray, Bruce Witthuhn, Albert Barrios, Douglas Yee. Detection of insulin receptor isoforms by mass spectrometry in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-03-02.

Severe Hepatic Insulin Resistance Induces Vascular Dysfunction: Improvement by Liver-Specific Insulin Receptor Isoform A Gene Therapy in a Murine Diabetic Model.

Background: Cardiovascular dysfunction is linked to insulin-resistant states. In this paper, we analyzed whether the severe hepatic insulin resistance of an inducible liver-specific insulin receptor knockout (iLIRKO) might generate vascular insulin resistance and dysfunction, and whether insulin receptor (IR) isoforms gene therapy might revert it. Methods: We studied in vivo insulin signaling in aorta artery and heart from iLIRKO. Vascular reactivity and the mRNA levels of genes involved in vascular dysfunction were analyzed in thoracic aorta rings by qRT-PCR. Finally, iLIRKO mice were treated with hepatic-specific gene therapy to analyze vascular dysfunction improvement. Results: Our results suggest that severe hepatic insulin resistance was expanded to cardiovascular tissues. This vascular insulin resistance observed in aorta artery from iLIRKO mice correlated with a reduction in both PI3K/AKT/eNOS and p42/44 MAPK pathways, and it might be implicated in their vascular alterations characterized by endothelial dysfunction, hypercontractility and eNOS/iNOS levels’ imbalance. Finally, regarding long-term hepatic expression of IR isoforms, IRA was more efficient than IRB in the improvement of vascular dysfunction observed in iLIRKO mice. Conclusion: Severe hepatic insulin resistance is sufficient to produce cardiovascular insulin resistance and dysfunction. Long-term hepatic expression of IRA restored the vascular damage observed in iLIRKO mice.

Tissue‐specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models

The two insulin receptor (IR) isoforms IR‐A and IR‐B are responsible for the pleiotropic actions of insulin and insulin‐like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR‐mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell‐type‐specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue‐dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes.

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.

Identification of Insulin Receptor Splice Variant B in Neurons by in situ Detection in Human Brain Samples

Insulin and its receptor are widely expressed in a variety of tissues throughout the body including liver, adipose tissue, liver and brain. The insulin receptor is expressed as two functionally distinct isoforms, differentiated by a single 12 amino acid exon. The two receptor isoforms, designated IR/A and IR/B, are expressed in a highly tissue and cell specific manner and relative proportions of the different isoforms vary during development, aging and disease states. The high degree of similarity between the two isoforms has prevented detailed studies as differentiation of the two isoforms by traditional immunological methods cannot be achieved. We describe here a new in situ RT-PCR/ FISH assay that allows for the visualization of IR/A and IR/B in tissue along with tissue specific markers. We used this new method to show for the first time that IR/A and IR/B are both expressed in neurons in the adult human brain. Thus, we present a method that enables the investigation of IR/A and IR/B insulin receptor isoform expression in situ in various tissues.

Insulin Receptor Isoforms in Physiology and Disease: An Updated View.

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.

Differential Expression of IR-A, IR-B and IGF-1R in Endometrial Physiology and Distinct Signature in Adenocarcinoma.

Type 2 diabetes and obesity are risk factors for endometrial hyperplasia and cancer, suggesting that hyperinsulinemia contributes to pathogenesis. Insulin action through insulin receptor (IR) splice variants IR-A and IR-B regulates cellular mitogenesis and metabolism, respectively. We hypothesized that IR-A and IR-B are differentially regulated in normal endometrium, according to mitogenic and metabolic requirements through the menstrual cycle, as well as in endometrial hyperplasia and cancer. IR-A, IR-B, and IGF-1 receptor (IGF-1R) mRNA was quantified in endometrium, endometrial epithelial and stromal cells, and in vitro after hormone stimulation. Academic center. Endometrium was collected from women with regular cycles (n = 71), complex hyperplasia (n = 5), or endometrioid adenocarcinoma (n = 11). In vitro sex-steroid treatment. IR-A and IR-B expression Results: IR-A increased dramatically during the early proliferative phase, 20-fold more than IR-B. In early secretory phase, IR-B and IGF-1R expression increased, reaching maximal expression, whereas IR-A decreased. In adenocarcinoma, IR-B and IGF-1R expression was 5- to 6-fold higher than normal endometrium, whereas IR-A expression was similar to IR-B. Receptor expression was unrelated to body mass index. IR-A was elevated during the normal proliferative phase, and in endometrial hyperplasia and adenocarcinoma. The dramatic early rise of IR-A in normal endometrium indicates IR-A is the predominant isoform responsible for initial estrogen-independent endometrial proliferation as well as that of cancer. IR-B is elevated during the normal secretory phase when glucose uptake and glycogen synthesis support embryo development. Differing from other cancers, IR-B expression equals mitogenic IR-A in endometrial adenocarcinoma. Differential IR isoform expression suggests a distinct role for each in endometrial physiology and cancer.

Insulin receptor isoform A ameliorates long-term glucose intolerance in diabetic mice.

ABSTRACTType 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte­-specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8 weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus.

Reduced insulin-like growth factor I receptor and altered insulin receptor isoform mRNAs in normal mucosa predict colorectal adenoma risk.

Hyperinsulinemia resulting from obesity and insulin resistance is associated with increased risk of many cancers, but the biology underlying this risk is unclear. We hypothesized that increased mRNA levels of the insulin-like growth factor I receptor (IGFIR) versus the insulin receptor (IR) or elevated ratio of IR-A:IR-B isoforms in normal rectal mucosa would predict adenoma risk, particularly in individuals with high body mass index (BMI) or plasma insulin. Biopsies from normal rectal mucosa were obtained from consenting patients undergoing routine colonoscopy at University of North Carolina Hospitals (Chapel Hill, NC). Subjects with colorectal adenomas were classified as cases (n = 100) and were matched to adenoma-free controls (n = 98) based on age, sex, and BMI. IGFIR and IR mRNA levels were assessed by qRT-PCR, and IR-A:IR-B mRNA ratios by standard PCR. Plasma insulin and crypt apoptosis were measured by ELISA and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. Logistic regression models examined relationships between receptor mRNAs, BMI, plasma insulin, and adenoma risk. Unexpectedly, cases were significantly more likely to have lower IGFIR mRNA levels than controls. No overall differences in total IR mRNA or IR-A:IR-B ratios were observed between cases and controls. Interestingly, in patients with high plasma insulin, increased IR-A:IR-B ratio was associated with increased likelihood of having adenomas. Our work shows novel findings that reduced IGFIR mRNA and, during high plasma insulin, increased IR-A:IR-B ratios in normal rectal mucosa are associated with colorectal adenoma risk. Our work provides evidence supporting a link between IGFIR and IR isoform expression levels and colorectal adenoma risk.

Insulin receptor isoform expression in ovarian cancer in African American women

Objectives: Two insulin receptor (INSR) isoforms, (INSR-B) and a splice variant that signals for cell proliferation and survival (INSR-A), are expressed in ovarian cancer (OC) cell lines. Obesity and type 2 diabetes are more common in the African American (AA) population, but little is known about OC INSR isoform expression in this group of patients. We hypothesized that differential expression of INSR-A in OC from AA women could represent a therapeutic target in this population.

Increased IR-A/IR-B ratio in non-small cell lung cancers associates with lower epithelial-mesenchymal transition signature and longer survival in squamous cell lung carcinoma

BackgroundTo evaluate the insulin receptor isoform mRNA expression status in non-small cell lung cancer (NSCLC) patients.MethodsRNA-seq data from 614 NSCLC [355 adenocarcinomas (LUAD) and 259 squamous cell carcinomas (LUSC)] and 92 normal lung specimens were obtained from The Cancer Genome Atlas (TCGA) to evaluate the mRNA expression of insulin receptor isoform A (IR-A) and insulin receptor isoform B (IR-B). The differential expression status of the insulin receptor isoforms in NSCLC patients was confirmed using qRT-PCR assays with lung cancer cDNA arrays and primary tumor samples.ResultsThe mRNA expression levels of IR-B were significantly lower in some NSCLC samples compared to normal lung specimens, including both LUAD and LUSC. Notably, no IR-B transcripts were detected - only the IR-A isoform was expressed in 11% of NSCLC patients. This decrease in IR-B expression contributed to an elevated IR-A/IR-B ratio, which was also associated with lower epithelial-mesenchymal transition gene signatures in NSCLC and longer patient survival under standard of care in LUSC. In addition to NSCLC, RNA-seq data from TCGA revealed a similar increase in IR-A/IR-B ratio in many other cancer types, with high prevalence in acute myeloid leukemia, glioblastoma multiforme, and brain lower grade glioma.ConclusionsOur results indicate a common reduction of the mRNA expression level of IR-B and an increased IR-A/IR-B mRNA ratio in NSCLC and other tumor types. The relationship of altered IR-A/IR-B ratios with cancer progression and patient survival should be prospectively explored in future studies.

Overexpression of the Insulin Receptor Isoform A Promotes Endometrial Carcinoma Cell Growth

Epidemiological studies have demonstrated that type 2 diabetes mellitus (T2DM) and hyperinsulinemia are associated closely with endometrial carcinoma risk, although the molecular mechanism remains unclear. Insulin receptor isoformA expression is upregulated in many cancer cells and tissues, which suggests that IR-A-mediated signaling pathways may have important implications for cancer pathogenesis. We measured the expression of insulin receptor isoforms (IR-A and IR–B in the normal endometrium tissues, the endometrial carcinoma tissues and the endometrial carcinoma cell lines. We found that the total insulin receptor (IR) and IR-A expression mRNA levels and the ratio of IR-A to total IR in endometrial carcinoma specimens were significantly higher than them in control endometrial tissue specimens(P<0.05). Further analysis indicated that the tendency was more prominently in patients with T2DM. IR-A mRNA was differentially expressed in four endometrial carcinoma cell lines (Ishikawa, KLE, RL95-2 and HEC-1-A. RL95-2 cells have a low endogenous IR-A expression, and these were used to construct a stable cell line overexpressing IR-A. We found that IR-A overexpression significantly increased cell proliferation, the proportion of cells in S phase, activation of the Akt pathway and tumorigenicity of xenografts in nude mice. In contrast, there was no significant difference in the the percentage of apoptotic cells between cells overexpressing IR-A and control cells. Moreover, levels of phosphorylated ERK1/2 protein were significantly decreased in cells overexpressing IR-A relative to controls. These findings reveal the pivotal role of IR-A in endometrial cancer carcinogenesis, and suggest that the association of elevated IR-A levels with cell proliferation and tumorigenicity may be causally linked to its effect on the proportion of cells in S phase and the activation of the Akt pathway.

Abstract 5268: Differential expression of insulin receptor isoforms in human lung cancer.

Abstract Substantial population and preclinical data have indicated that the insulin growth factor (IGF) pathway plays an important role in lung cancer. Although results from early clinical trials targeting the IGF1R showed some evidence of response in non-small cell lung cancer (NSCLC), larger randomized phase III trials have not shown clear clinical benefit in combination with conventional strategies. These disappointing results have focused attention on the insulin receptor isoform A (IR-A) as one intrinsic resistance factor to anti-IGF1R therapy due to its ability to mediate growth-promoting signaling through binding to IGF-II. However, the expression status of insulin-receptor isoforms in lung cancer and its subtypes is largely unknown. In order to address this issue, the mRNA expression levels of insulin-receptor isoforms were studied in 50 normal lung samples and 183 lung cancer samples from complementary DNA (cDNA) arrays. Additionally, 31 primary small cell lung cancer (SCLC) tumor samples with paired adjacent normal tissues were also evaluated. The mRNA expression levels of insulin receptor isoform B (IR-B) were significantly lower in NSCLC than normal lung samples (n=176, p &amp;lt; 0.001), including adenocarcinoma (n=85, p &amp;lt; 0.0001), squamous cell carcinoma (n=60, p &amp;lt; 0.0001) and large cell lung cancer samples (n=16, p &amp;lt; 0.0001). The mRNA expression levels of IR-B were also significantly lower in SCLC primary tumor samples (P=0.002). The mRNA levels for IR-A were significantly higher in primary SCLC samples (p &amp;lt; 0.02) than paired adjacent normal, but no significant differences between normal lung and NSCLC or its subtypes were observed (p &amp;gt; 0.05). When comparing tumor to normal lung samples, the IR-A:IR-B mRNA expression ratios were significantly higher in primary SCLC samples (p &amp;lt;0.0001). The IR-A:IR-B mRNA expression ratios were also significantly higher in all NSCLC samples compared to normal lung, (p &amp;lt; 0.001) including adenocarcinoma (p &amp;lt; 0.0001), squamous cell carcinoma (p &amp;lt; 0.0001) and large-cell lung cancer (p &amp;lt; 0.0001). Our results indicated that the reduction of the level of IR-B expression and an increase in the IR-A:IR-B expression ratio may highlight patients with active IR-A signaling, and may be more informative than identifying those with increased IR-A expression only. Additionally, these results suggested that an increased IR-A:IR-B expression ratio might be a useful biomarker in selecting patients that may have an intrinsic resistance to anti-IGF1R therapy in lung cancer. Citation Format: Jiaqi Huang, Katie Streicher, Chris Morehouse, Kim Lehmann, Philip Brohawn, Wei Zhu, Meggan Czapiga, Theresa LaVallee, Karen Mitz, Laura Richman, Bahija Jallal, Yihong Yao. Differential expression of insulin receptor isoforms in human lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2013-5268

Preliminary investigation of the expression and functions of insulin receptor isoforms in endometrial carcinoma

To investigate the expression of insulin receptor isoforms (IR, including IR-A and IR-B) in endometrial carcinoma (EC), and explore the role of IR-A in the growth of endometrial carcinoma cells. The expression of IR isoforms were detected by reverse transcription (RT)-PCR and real-time PCR in 4 different endometrial cancer cell lines (HEC-1-A, Ishikawa, RL95-2 and KLE), with human breast cancer cell line MCF-7 cells and hepatocellular carcinoma cell line Hep-G2 as positive control and in the endometrial cancer tissue specimens of 42 cases, admitted in Peking University People's Hospital from November 2007 to July 2009, with normal endometrial tissues from 15 cases of ovarian neoplasms at the same period as controls. The relationships among the expression of IR, IR-A isoforms and the clinicopathological parameters of EC tissues were analyzed by Spearman rank correlation analysis. An eukaryotic IR-A expression plasmid was constructed and transfected into RL95-2 cells [RL95-2(IR-A)] for overexpression of IR-A in RL95-2 cells, in which the expression of IR-A originally was low. Non radioactive cell proliferation assay-MTS was used to determine the proliferation curves in the four EC cells listed above and in RL95-2 or RL95-2 (IR-A). (1) There were two isoforms of IR-A and IR-B co-expressed were detected in EC cells and EC tissues. Among four kinds of EC cell lines, the expression level of IR mRNA in RL95-2 cells was the highest, followed by Ishikawa, KLE and HEC-1-A cells, in which the relative IR mRNA expression levels were (26.54 ± 1.82)×10(-4), (15.44 ± 3.29)×10(-4), (10.14 ± 0.10)×10(-4) and (2.63 ± 0.23)×10(-4), respectively (P < 0.01). The expression level of IR-A mRNA was the highest in Ishikawa cells, followed by KLE, RL95-2 and HEC-1-A cells, with the relative expression levels were (12.07 ± 3.31)×10(-4), (4.68 ± 0.63)×10(-4), (3.03 ± 0.22)×10(-4) and(1.46 ± 0.03)×10(-4), respectively (P < 0.01). The relative expression level of IR and IR-A mRNA were 0.017 ± 0.013 and 0.011 ± 0.010 in the EC tissues, respectively, compared with 0.015 ± 0.014 and 0.010 ± 0.012 in the controls, in which there were no significant differences in the expression level of IR or IR-A mRNA between EC tissues and the control (P = 0.662, P = 0.780). The expression of IR and IR-A in EC tissues had no significant relevance with International Federation of Gynecology and Obstetrics (FIGO) stage, cell differentiation, depth of myometrial invasion, invasion of lymph-vascular space, lymph nodes metastasis, the expression status of estrogen receptor, human progesterone receptor, and PTEN gene (all P > 0.05). The expression of IR-A mRNA in EC patients with type 2 diabetes mellitus (DM) was significantly higher than that in patients without type 2 DM (P = 0.031), while there were no statistical correlation between the expression of IR mRNA and type 2 DM (P = 0.438). (2) The proliferation rates of the four kinds of EC cells was positively related with the IR-A expression ratio, with the most growth potential in Ishikawa, followed by HEC-1-A, KLE and RL95-2 cells. The overexpression of IR-A in RL95-2 (IR-A) cells showed a significant proliferation-promoting effect than that in control RL95-2 cells (P < 0.01). There are two isoforms of IR-A and IR-B co-expressed in EC. The overexpression of IR-A may promote the proliferation of EC cells.

Quantifying insulin receptor isoform expression in FFPE breast tumors

BackgroundThe development of predictive biomarkers for IGF targeted anti-cancer therapeutics remains a critical unmet need. The insulin receptor A isoform (InsR-A) has been identified as a possible biomarker candidate but quantification of InsR-A in widely available formalin fixed paraffin embedded (FFPE) tissues is complicated by its similarities with the metabolic signaling insulin receptor isoform B (InsR-B). In the present study, qPCR based assays specific for InsR-A, InsR-B and IGF-1R were developed for use in FFPE tissues and tested for feasible use in clinical archived FFPE estrogen receptor (ER)+and ER− breast cancer tumors. DesignFFPE compatible primer sets were designed with amplicon sizes of less than 60 base pairs and validated for target specificity, assay repeatability and amplification efficiency. FFPE tumors from ER+ (n=83) and ER−(n=64) primary untreated breast cancers, and ER+ hormone refractory (HR ER+) (n=61) breast cancers were identified for feasibility testing. The feasible use of InsR-A and InsR-B qPCRs were tested using all tumor groups and the feasibility of IGF-1R qPCR was determined using HR ER+ tumors. ResultsAll qPCR assays were highly reproducible with amplification efficiencies between 96-104% over a 6 log range with limits of detection of 4 or 5 copies per reaction. Greater than 90% of samples were successfully amplified using InsR-A, InsR-B or IGF-1R qPCR primer sets and greater than 88% of samples tested amplified both InsR isoforms or both isoforms and IGF-1R. InsR-A was the predominant isoform in 82% ER+, 68% ER− and 100% HR ER+ breast cancer. Exploratory analyses demonstrated significantly more InsR-A expression in ER+ and HR ER+ groups compared to InsR-B (ER+ p<0.05, HR ER+ p<0.0005) and both groups had greater InsR-A expression when compared to ER− tumors (ER+ p<0.0005, HR ER+ p<0.05). IGF-1R expression of HR ER+ tumors was lower than InsR-A (p<0.0005) but higher than InsR-B (p<0.0005). The InsR-B expression of HR ER+ tumors was significantly reduced compared other tumor subgroups (ER+ and ER−, p<0.0005) and lead to a significant elevation of HR ER+ InsR-A: InsR-B ratios (ER+ and ER−, p<0.0005). ConclusionsThe validated, highly sensitive InsR-A and InsR-B qPCR based assays presented here are the first to demonstrate the feasible amplification of InsR isoforms in FFPE tissues. Quantification data generated from this feasibility study indicating InsR-A is more predominant than InsR-B in breast cancer support the use of these assays for further investigation of InsR-A and InsR-B as predictive biomarkers for IGF targeted therapeutics.

Differential expression of insulin receptor isoforms A and B in highly proliferative stem and tumor cells vs. differentiated intestinal epithelial cells

Little is known about expression and roles of insulin receptor (IR) in the intestinal epithelium despite emerging links between obesity, hyperinsulinemia, and intestinal dysfunction or cancer risk. IR exists as an IR‐B isoform that mediates insulin's metabolic actions and an IR‐A isoform implicated in fetal growth and recently, cancer. This study used Sox9‐EGFP reporter mice, ApcMin/+ mice, and human colorectal cancer cell lines (CRC) to test the hypothesis that IR‐A and IR‐B have distinct expression profiles in highly proliferative vs differentiated normal intestinal epithelial cells (IEC) or CRC.ResultsIR‐A predominates in highly proliferative intestinal epithelial stem cells and progenitors, while IR‐B predominates in differentiated lineages. In ApcMin/+ mice, the IR‐B:IR‐A ratio is dramatically decreased in tumor vs non‐tumor tissue in small intestine (0.65 ± 0.02 vs 1.3 ± 0.03) and colon (0.63 ± 0.1 vs 1.27 ± 0.9). The IR‐B:IR‐A ratio is higher in CRC with the ability to differentiate vs undifferentiated, aggressively growing CRC. The ratio of IR‐B:IR‐A is significantly increased in differentiated vs undifferentiated Caco2 cells (2.2 ± 0.2 vs 1.5 ± 0.1). We conclude that relative expression levels of IR‐B:IR‐A may be critical determinants of normal IEC or CRC differentiation. Grants: F31AG040943 (SFB) and NIH DK040247‐19 (PKL).

Altered Expression of Insulin Receptor Isoforms in Breast Cancer

PurposeInsulin-like growth factor (IGF) signaling through human insulin receptor isoform A (IR-A) contributes to tumorigenesis and intrinsic resistance to anti-IGF1R therapy. In the present study, we (a) developed quantitative TaqMan real time-PCR-based assays (qRT-PCR) to measure human insulin receptor isoforms with high specificity, (b) evaluated isoform expression levels in molecularly-defined breast cancer subtypes, and (c) identified the IR-A:IR-B mRNA ratio as a potential biomarker guiding patient stratification for anti-IGF therapies.Experimental DesignmRNA expression levels of IR-A and IR-B were measured in 42 primary breast cancers and 19 matched adjacent normal tissues with TaqMan qRT-PCR assays. The results were further confirmed in 165 breast cancers. The tumor samples were profiled using whole genome microarrays and subsequently subtyped using the PAM50 breast cancer gene signature. The relationship between the IR-A:IR-B ratio and cancer subtype, as well as markers of proliferation were characterized.ResultsThe mRNA expression levels of IR-A in the breast tumors were similar to those observed in the adjacent normal tissues, while the mRNA levels of IR-B were significantly decreased in tumors. The IR-A:IR-B ratio was significantly higher in luminal B breast cancer than in luminal A. Strong concordance between the IR-A:IR-B ratio and the composite Oncotype DX proliferation score was observed for stratifying the latter two breast cancer subtypes.ConclusionsThe reduction in IR-B expression is the key to the altered IR-A:IR-B ratio observed in breast cancer. The IR-A:IR-B ratio may have biomarker utility in guiding a patient stratification strategy for an anti-IGF therapeutic.

Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

OBJECTIVETo determine whether insulin reverses gestational diabetes mellitus (GDM)–reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs).RESEARCH DESIGN AND METHODSPrimary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine1177 phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist).RESULTSUmbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO–dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME.CONCLUSIONSGDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM.