Insulin Growth Factor Pathway Research Articles

Abstract P3-07-22: Presurgical Trial of Metformin plus Atorvastatin in Women with Operable Breast Cancer

Abstract Introduction: Metformin is an oral anti-diabetic agent that exhibits direct anti-proliferative effects on pre-clinical models through activation of the AMPK pathway. However, pre-surgical studies of metformin alone among women with operable breast cancer have not consistently shown reductions in tumor proliferation, and adjuvant metformin in women with high-risk operable breast cancer did not improve invasive disease-free survival compared with placebo. Dysregulation of the mevalonate pathway of cholesterol synthesis can also lead to cell proliferation, and inhibition of HMG-CoA reductase by statins can decrease tumor proliferation. There is close interaction between the AMPK and mevalonate pathways, and dual therapy with a statin and metformin might be synergistic to decrease cell proliferation. We evaluated the effect of combination therapy with metformin plus atorvastatin on markers of proliferation (i.e. Ki67 proliferation index) in women with operable breast cancer. Methods: We conducted an open-label, single-arm presurgical “window of opportunity” study of metformin plus atorvastatin in non-diabetic women age 21+ years with newly-diagnosed stage 0-III operable breast cancer at Columbia University Irving Medical Center (CUIMC). Enrolled patients received metformin 1500mg oral [p.o.] daily (500mg in the morning/1000mg in the afternoon) and atorvastatin 80mg p.o. nightly for up to 4 weeks before breast surgery. The primary endpoint was change in Ki67 proliferation index from baseline (diagnostic biopsy) to post-treatment (surgical specimen). Secondary endpoints included change in body mass index (BMI), waist and hip circumferences, tumor assessment of AMPK/mTOR signaling and apoptosis, and reduction of fasting markers of the insulin growth factor pathway. Paired t-tests were conducted to assess difference in ln(Ki67) pre- and post-therapy, as well as differences in absolute Ki67, BMI, and waist/hip circumferences pre- and post-therapy, at a level of significance of 0.05. Results: Between Nov. 2013 and Jan. 2018, 22 women were enrolled, and two withdrew consent prior to study treatment. Among evaluable participants (n=20), 45% were Hispanic with median age 56 years (range, 33-73) and median baseline BMI 28.4 kg/m2 (range, 22.5-45.8). All had hormone receptor-positive (HR+), HER2-negative breast cancer, and 16 (80%) had invasive cancer. Median time on study treatment was 11 days (range, 5-29). Changes in Ki67 and anthropomorphic measures are shown in Table 1. There was no significant change in BMI, waist or hip circumference with study treatment. Among women with available Ki67 measurements (n=11), there was no significant difference in pre- and post-treatment ln(Ki67) (p=0.25). There was a numeric decrease in absolute Ki67, though statistical significance was not reached (p=0.09). Discussion: There was a numeric reduction in absolute Ki-67 with presurgical metformin plus atorvastatin in patients with newly diagnosed HR+/HER2- breast cancer, although our analysis was limited by small sample size and statistical significance was not achieved. There was no difference in ln(Ki67) or anthropometric measurements. Analyses of additional tissue and serum biomarkers, including markers of insulin resistance, are ongoing to identify associations with absolute Ki67 Table 1 Citation Format: Julia E. McGuinness, Katherine D. Crew, Meghna S. Trivedi, Melissa K. Accordino, Shing M. Lee, Hua Guo, Hanina Hibshoosh, Dawn Hershman, Kevin Kalinsky. Presurgical Trial of Metformin plus Atorvastatin in Women with Operable Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-22.

Gene expression associated with lymphovascular invasion and genomic risk in early-stage breast cancer.

559 Background: Lymphovascular invasion (LVI), the passage of carcinoma cells through lymphatic and blood vessels, is an important early step in metastasis; however, LVI is excluded from most breast cancer (BC) clinical risk assessments. Previous studies assessed the prognostic value of LVI to estimate clinical outcomes. To gain understanding of the molecular basis of LVI, we evaluated differentially expressed genes (DEGs) between tumors with LVI versus those without LVI, stratified by the 70-gene signature (MammaPrint/MP) and 80-gene molecular subtyping signature (BluePrint/BP). Methods: The prospective, observational FLEX Study (NCT03053193) includes stage I-III BC patients who receive MP/BP testing and consent to full transcriptome and clinical data collection. Patients with LVI (n=581) and without LVI (n=600, randomly selected), enrolled from 2017 to present, were included. LVI was assessed by local pathology laboratories. Differential gene expression analysis of 44k Agilent microarray data was performed with R limma package. DEGs were compared within all samples, BP Luminal subtype, MP risk groups (Low Risk [LR]/Luminal A and High Risk [HR]/Luminal B), and by lymph node (LN) status. DEGs with FDR<0.05 were considered significant. Results: Of tumors with LVI (LVI+), 66% were MP HR; notably, 51% of tumors without LVI (LVI-) were MP HR. LVI was associated with larger T stage, LN involvement, high grade, negative ER status by IHC, and younger patient age (LVI+ vs. LVI-, p<0.05 for all comparisons). Patient ethnicity, obesity, and tumor type did not differ by LVI status; however, prevalence of type 2 diabetes trended higher in patients with LVI+ HR tumors (21%), compared with LVI- HR (15%, p=0.09) and LVI+ LR (11%, p=0.004). There were significant transcriptomic differences between LVI+ and LVI, with most DEGs evident in the Luminal B subset. DEGs in LVI+, LN-negative (LN-) tumors overlapped substantially with the overall Luminal group analysis. Functional enrichment analysis showed dysregulation of cell cycle, extracellular matrix (ECM) organization, cell adhesion, and cytokine receptor pathways. Gene sets related to insulin growth factor pathways were also enriched in LVI+ tumors. Conclusions: DEGs associated with LVI were primarily found in MP HR Luminal, LN-negative tumors; enrichment analysis suggested dysregulation of ECM organization and cell adhesion pathways, consistent with previous reports. DEGs were not associated with LVI presence in LN+ tumors, suggesting that LVI assessment may be less relevant in LN+ breast cancer. Future studies will assess clinical outcomes, as well as LVI-associated gene expression in BP Basal- and HER2-type tumors. However, the current analysis indicates few DEGs in LVI+ MP LR tumors; thus, the potential prognostic information gained from LVI-associated gene expression is likely already captured by the MP and BP signatures. Clinical trial information: NCT03053193.

Enhancer Reprogramming Confers Dependence on Glycolysis and IGF Signaling in KMT2D Mutant Melanoma

SUMMARYHistone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors.

IGF1R activation and the in vitro antiproliferative efficacy of IGF1R inhibitor are inversely correlated with IGFBP5 expression in bladder cancer

BackgroundThe insulin growth factor (IGF) pathway has been proposed as a potential therapeutic target in bladder cancer. We characterized the expression of components of the IGF pathway — insulin growth factor receptors (INSR, IGF1R, IGF2R), ligands (INS, IGF1, IGF2), and binding proteins (IGFBP1–7, IGF2BP1–3) — in bladder cancer and its correlation with IGF1R activation, and the anti-proliferative efficacy of an IGF1R kinase inhibitor in this setting.MethodsWe analyzed transcriptomic data from two independent bladder cancer datasets, corresponding to 200 tumoral and five normal urothelium samples. We evaluated the activation status of the IGF pathway in bladder tumors, by assessing IGF1R phosphorylation and evaluating its correlation with mRNA levels for IGF pathway components. We finally evaluated the correlation between inhibition of proliferation by a selective inhibitor of the IGF1R kinase (AEW541), reported in 13 bladder cancer derived cell lines by the Cancer Cell Line Encyclopedia Consortium and mRNA levels for IGF pathway components.ResultsIGF1R expression and activation were stronger in non-muscle-invasive than in muscle-invasive bladder tumors. There was a significant inverse correlation between IGF1R phosphorylation and IGFBP5 expression in tumors. Consistent with this finding, the inhibition of bladder cell line viability by IGF1R inhibitor was also inversely correlated with IGFBP5 expression.ConclusionThe IGF pathway is activated and therefore a potential therapeutic target for non muscle-invasive bladder tumors and IGFBP5 could be used as a surrogate marker for predicting tumor sensitivity to anti-IGF therapy.

Collaborative rewiring of the pluripotency network by chromatin and signalling modulating pathways

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) represents a profound change in cell fate. Here, we show that combining ascorbic acid (AA) and 2i (MAP kinase and GSK inhibitors) increases the efficiency of reprogramming from fibroblasts and synergistically enhances conversion of partially reprogrammed intermediates to the iPSC state. AA and 2i induce differential transcriptional responses, each leading to the activation of specific pluripotency loci. A unique cohort of pluripotency genes including Esrrb require both stimuli for activation. Temporally, AA-dependent histone demethylase effects are important early, whereas Tet enzyme effects are required throughout the conversion. 2i function could partially be replaced by depletion of components of the epidermal growth factor (EGF) and insulin growth factor pathways, indicating that they act as barriers to reprogramming. Accordingly, reduction in the levels of the EGF receptor gene contributes to the activation of Esrrb. These results provide insight into the rewiring of the pluripotency network at the late stage of reprogramming.

The oestrogen pathway underlies the evolution of exaggerated male cranial shapes in Anolis lizards.

Sexual dimorphisms vary widely among species. This variation must arise through sex-specific evolutionary modifications to developmental processes. Anolis lizards vary extensively in their expression of cranial dimorphism. Compared with other Anolis species, members of the carolinensis clade have evolved relatively high levels of cranial dimorphism; males of this clade have exceptionally long faces relative to conspecific females. Developmentally, this facial length dimorphism arises through an evolutionarily novel, clade-specific strategy. Our analyses herein reveal that sex-specific regulation of the oestrogen pathway underlies evolution of this exaggerated male phenotype, rather than the androgen or insulin growth factor pathways that have long been considered the primary regulators of male-biased dimorphism among vertebrates. Our results suggest greater intricacy in the genetic mechanisms that underlie sexual dimorphisms than previously appreciated.

Early alterations in energy metabolism in the hippocampus of APPswe/PS1dE9 mouse model of Alzheimer's disease

The present study had focused on the behavioral phenotype and gene expression profile of molecules related to insulin receptor signaling in the hippocampus of 3 and 6month-old APPswe/PS1dE9 (APP/PS1) transgenic mouse model of Alzheimer's disease (AD). Elevated levels of the insoluble Aβ (1–42) were detected in the brain extracts of the transgenic animals as early as 3months of age, prior to the Aβ plaque formation (pre-plaque stage). By the early plaque stage (6months) both the soluble and insoluble Aβ (1–40) and Aβ (1–42) peptides were detectable. We studied the expression of genes related to memory function (Arc, Fos), insulin signaling, including insulin receptor (Insr), Irs1 and Irs2, as well as genes involved in insulin growth factor pathways, such as Igf1, Igf2, Igfr and Igfbp2. We also examined the expression and protein levels of key molecules related to energy metabolism (PGC1-α, and AMPK) and mitochondrial functionality (OXPHOS, TFAM, NRF1 and NRF2). 6month-old APP/PS1 mice demonstrated impaired cognitive ability, were glucose intolerant and showed a significant reduction in hippocampal Insr and Irs2 transcripts. Further observations also suggest alterations in key cellular energy sensors that regulate the activities of a number of metabolic enzymes through phosphorylation, such as a decrease in the Prkaa2 mRNA levels and in the pAMPK (Thr172)/Total APMK ratio. Moreover, mRNA and protein analysis reveals a significant downregulation of genes essential for mitochondrial replication and respiratory function, including PGC-1α in hippocampal extracts of APP/PS1 mice, compared to age-matched wild-type controls at 3 and 6months of age. Overall, the findings of this study show early alterations in genes involved in insulin and energy metabolism pathways in an APP/PS1 model of AD. These changes affect the activity of key molecules like NRF1 and PGC-1α, which are involved in mitochondrial biogenesis. Our results reinforce the hypothesis that the impairments in both insulin signaling and energy metabolism precede the development of AD amyloidogenesis.

A phase I trial of cixutumumab (C) (IMC-A12) and sorafenib (S) for treatment of advanced hepatocellular carcinoma (HCC).

293 Background: The insulin growth factor (IGF) pathway is activated in hepatocarcinogenesis. C is a monoclonal antibody against human insulin-like growth factor-1 receptor (IGF-1R). In vivo HCC models show activated IGF signaling and antitumor effects due to C. Given the cross-talk between the IGF and VEGF pathways, the combination of C and S was chosen for study. Methods: This study evaluated the safety of C (2, 4 or 6 mg/kg) IV weekly with standard doses of S (400 mg po bid) in patients (pts) with HCC without standard curative options. Eligibility criteria included: no prior systemic therapy, Child-Pugh score of A or B7, ECOG 0 or 1, platelets > 75,000/mm3, and albumin > 2.8 g/dl. The study used a standard 3+3 design. One cycle was 28 days. Results: A total of 21 pts (17 males and 4 females) were enrolled; mean age was 63 years (43-85); 10 Asian, 4 Hispanic, 5 White, 1 Black, and 1 Native American. There were 3 dose limiting toxicities (DLTs); grade 3 hyperglycemia, grade 3 hypophosphatemia, and grade 5 peritonitis (table). The maximum tolerated dose (MTD) was C 4 mg/Kg and S 400 mg BID. Eighteen of 21 (86%) pts had ≥ grade 3 toxicities attributable to treatment. Grade 3 adverse events that occurred in ≥ 2 pts were: diarrhea (4; 19%), hypertension (4; 19%), thrombocytopenia (3; 14%), palmar-plantar erythrodysesthesia (2; 10%), hyperglycemia (2; 10%), and fatigue (2; 10%). There was one grade 4 colonic perforation and one grade 5 peritoneal infection in the same pt. The median number of cycles completed was 4 (0-19 cycles). Sixteen of the 21 pts completed 2 cycles and were evaluated for response. Thirteen of the sixteen (81%) (95% CI: 54%-96%) achieved stable disease. The median event-free survival was 3.8 months (95%CI: 1.9, 11.3). The median OS was 13.1 months (95% CI: 7.6, undefined). Conclusions: The majority of adverse events in pts treated with the combination of C and S were typical of sorafenib toxicity. There was preliminary evidence of efficacy as seen in the median OS. Analysis of biomarker correlative data is on-going. Clinical trial information: NCT01008566. [Table: see text]

Abstract OT2-6-06: Pre-surgical “window of opportunity” trial of metformin and atorvastatin in newly diagnosed operable breast cancer

Abstract Background: Breast cancer requires energy homeostasis shifts with enhanced anabolism to enable rapid growth and continued proliferation. The main energy regulatory system is the AMP-activated kinase (AMPK) pathway triggered by changes in the AMP/ATP ratio. AMPK pathway closely interacts with the PI3K/AKT signaling pathway, with both pathways affecting downstream function of the master regulator mTOR. “Window of opportunity” studies with metformin alone, an AMPK inhibitor, have resulted in mixed results in reducing tumor proliferation in women with early-stage operable breast cancer. Reduction in tumor proliferation has been demonstrated with statins alone (i.e. HMG CoA reductase inhibitors) in pre-surgical trials. Dual therapy with both metformin and atorvastatin demonstrate synergistic activity in preclinical studies in cancer cell lines, showing enhanced anti-proliferative effect. The purpose of this study is to determine the effects of dual therapy with metformin and atorvastatin in women with newly diagnosed BC between breast biopsy and surgery. Trial Design: Patients (n = 40) will receive metformin 1500mg (500 mg am/1000 mg pm) and atorvastatin 80mg pm, for 2-4 weeks following a diagnostic biopsy and prior to surgery (goal: at least 2 weeks). The main eligibility criteria for this open-label, single-institution, pre-surgical trial include operable stage 0-III BC. Patients must have at least 1 cm of tumor based on palpation or imaging to ensure sufficient pre-treatment tissue. Patients not considered for neoadjuvant chemotherapy are eligible. Specific Aims: Our hypothesis is that pre-surgical metformin plus atorvastatin will result in a significant decrease in the tumor proliferation marker Ki-67. Ki-67 will be log-transformed ln(ki-67), per international guidelines. Secondary objectives include evaluation of functional proteomic changes, such as AMPK/mTOR pathway signaling and apoptosis, by reverse phase protein array (RPPA), as well as assessment of changes in serum insulin, lipids, and markers of the insulin growth factor pathway. Statistical Methods: Paired t-tests will be calculated to assess modulations in ln(ki-67) before and after treatment. Compared to historical control, we will achieve 80% power with 40 patients, anticipating a -0.523 reduction of ln(ki-67) and standard deviation of 1.15 before and after metformin plus statin (significance level, p = 0.05). We will also be comparing changes in ln(ki-67) in the treated patients to historical controls matched by age, stage, and BMI, using a two-sample t-test at level 0.05. Frequency distributions and summary descriptive statistics will be calculated for all other biomarkers in the two groups. Correlations between all biomarkers and changes in Ki-67 proliferation marker will be analyzed in exploratory fashion. We will also explore differences in modulation of tumor proliferation and functional proteomics in grade III tumors as compared to other tumors. We anticipate accrual 2-3 patient/month, completing the trial within 18 months. Contact information: kk2693@columbia.edu. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT2-6-06.

Computational prediction of the PolyQ and CAG repeat spinocerebellar ataxia network based on sequence identity to untranslated regions

Computational prediction of biological networks would be a tremendous asset to systems biology and personalized medicine. In this paper, we use a moving window bioinformatic screen to identify transcripts with partial identity to the 5′ and 3′UTRs of the polyQ spinocerebellar ataxia (SCA) genes ATXN1, ATXN2, ATXN3, ATXN7, TBP and CACNA1A and the CAG repeat expansion gene PPP2R2B. We find that the bioinformatic screen enriches for transcripts that encode proteins that interact and that have functions relevant to polyQ SCA. Transcription control and RNA binding are the primary functional groups represented in the proteins from the combined screens. The insulin growth factor pathway, the WNT pathway, long term potentiation, melanogenesis and ATM mediated DNA repair pathways were identified as important pathways. UGUUU repeats were identified as an abundant motif in the SCA network and PAXIP1, CELF2, CREBBP, EBF1, PLEKHG4, SRSF4, C5orf42, NFIA, STK24, and YWHAG were identified as statistically significant proteins in the polyQ and PPP2R2B network.

Patterns of deregulation of insulin growth factor signalling pathway in paediatric and adult gastrointestinal stromal tumours

BackgroundData regarding the patterns and the mechanisms of deregulation of the insulin growth factor (IGF) pathway in adult and paediatric gastrointestinal stromal tumours (GISTs) are limited. MethodsWe investigated the expression profiling of the genes encoding the main components of the IGF signalling pathway in 131 GISTs (106 adults, 21 paediatric and four young adults) and 25 other soft-tissue sarcomas (STS) using an Affymetrix U133A platform. IGF2 was investigated for loss of imprinting (LOI) whereas IGF1R was analysed for copy number aberration and mutation. ResultsIGF2 was the most highly overexpressed gene of the IGF pathway in GIST. IGF2 expression was also significantly higher than in other STS. IGF2 expression was correlated to the age onset and mutational status of GIST. Indeed, IGF2 expression was significantly higher in the ‘adult’ group than in the ‘paediatric’ and ‘young adult’ groups. Among adult GIST, IGF2 expression was higher in tumours lacking Homo sapiens v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) or alpha-type platelet-derived growth factor receptor (PDGFRA) mutations in comparison with mutated cases. A trend for a higher expression of IGF2 in resistant GIST in comparison to responsive GIST was also found. Overexpression of IGF2 was not related to LOI. Conversely, the expression of the IGF1R gene was significantly higher in the paediatric group than in the adult group. No copy number gains or mutations of IGF1R were observed. ConclusionThe IGF pathway is deregulated in GIST with distinct patterns according to age onset and mutational status. The IGF pathway may represent a therapeutic target in patients with primary or secondary resistance to imatinib.

Abstract 1192: Regulation of CCND2, HMGA2, and the IGF pathway by LIN28B in HPV negative head and neck squamous cell carcinoma (HNSCC)

Abstract Background: Lin28 and Lin28B have recently been implicated in the post-transcriptional regulation of Let-7/miR-98 biogenesis. While Lin28B over-expression has been associated with worse clinical outcome in several human cancers, the prognostic significance of Lin28B expression and the molecular mechanism(s) by which it drives tumour progression remains largely unknown. Methods: Let-7-Resist Lin28B-expression plasmid was stably transfected into the FaDu human hypopharyngeal squamous cell carcinoma line. MicoRNA expression profiling was conducted using the Nanostring nCounter Analysis System. Genome-wide micro-array and in silico analyses were utilized to identify Let-7/miR-98 targets. Cell viability and apoptosis were measured using MTS assay and flow cytometry, respectively. In vivo experiments were conducted using SCID mice. Results: qRT-PCR analysis of genes involved in the miRNA biogenesis pathway (DICER, LIN28, LIN28B, and TUT4) suggested an inverse relationship between the expression of Lin28B and mature Let-7 miRNA. Over-expression of Lin28B mediated significant increase in the migration of FaDu cells in vitro, and tumor growth in SCID mice, which in turn, was associated with marked reduction in the Let-7 family of microRNAs. Using a tri-Pronged approach by combining prediction algorithms, experimentally-derived mRNA profiling, along with gene expression data in primary HNSCC samples identified several upregulated Let-7/miR98 gene targets in Lin28B-FaDu cells, including CCND2, HMGA2, and regulators of the insulin growth factor (IGF) mitogenic pathway. Concordantly, treating FaDu-Lin28B cells with IGF (10-50 nM) led to a 26% and 50% increase in cell viability under stress conditions (serum starvation), whereas treating FaDu-GFP cells with IGF had no effect on viability, suggesting the relevance of the IGF axis in FaDu-Lin28B cells. Furthermore, analyzing publicly available data sets on primary HPV positive vs. HPV negative HNSCC samples revealed a significantly higher expression of HMGA2, and genes that regulate the IGF pathway in the latter group of tumours, suggesting a possible role for Lin28B, Let-7/miR-98 and their downstream targets in mediating the more aggressive behavior of HPV negative HNSCC. Conclusions: Our data report for the first time, a potentially important role for Lin28B in regulating the IGF mitogenic pathway, as one possible mechanism in driving the biological behaviour of HPV negative HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1192. doi:10.1158/1538-7445.AM2011-1192

Gene Expression Profiling of Phenotypically-Defined Hormone-Receptor Positive Breast Cancer: Evidence for Increased Transcriptional Activity of the Insulin Growth Factor Receptor Pathway and Other Pathways.

Abstract Background: Approximately 70% of all breast cancers are hormone receptor (HR)-positive tumors that are sensitive to endocrine therapy, but some patients have recurrence despite adjuvant endocrine therapy. We performed an exploratory analysis of gene expression in HR-pos operable breast cancer in order to identify potential novel therapeutic targets and biomarkers associated with recurrence. Methods: RNA was extracted from primary tumor samples obtained from 776 patients with stage I-III breast cancer treated with adjuvant chemohormonal therapy in trial E2197 (JCO 2008; 26: 4092-4099), of whom 458 had HR-pos disease (defined in a central lab; JCO 2008; 26: 2473). We evaluated RNA expression patterns (by quantitative RT-PCR using a panel of 371 rationally selected genes) in HR-pos cases compared with the HR-neg cases using weighted T statistics, and determined which genes in the HR-pos, HER2-neg group were associated with recurrence (using Cox proportional hazards model score test, Korn's adjusted P value <5% with false discovery rate < 10%).Results: The top 10 genes exhibiting significantly higher expression in the HR-pos group (p≤ 6.17e-160) included ESR1 plus 5 estrogen regulated genes, confirming our approach of evaluating gene expression in phenotypically-defined subsets. Other pathways that exhibited higher expression in the HR-pos group (among the 40 top genes with higher expression, p<8.66e-53) included the insulin growth factor (IGF) (IRS1, IGFR1, IGFB2), Ras (RhoB, RhoC, RAB27B, GGPS1), and HER pathways (ERBB2, ERBB3, ERBB4), and other genes involved in apoptosis (BCL2, BCL2L1, BAG1, NME6, BBC3), signaling (MAPK3, SEMA3F, RXRA), mismatch repair (MSH3), cell cycle regulation (CCND1), stress response (HSPB1), and tumor suppressor genes (TP53BP1, APC). These patterns were similar in HER2-pos cases. Pathway analysis (Ingenuity) revealed substantial interconnectivity among these genes, especially between IGFR1, ERB2/3/4, MAPK3, BCL2, and CCND1, but not RhoB/RhoC. Genes for which increased expression was associated with increased recurrence included those associated with proliferation (TOP2A, AURKB, PLK1) and apoptosis (BIRC5 - survivin).Conclusions: This exploratory analysis reveals several pathways that exhibit higher transcriptional expression in HR-pos disease, some of which are also associated with a higher risk of recurrence, suggesting that they may be potential therapeutic targets. This provides rationale for testing agents currently available in the clinic that inhibit the IGF and other pathways. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5165.

Insulin Growth Factor Pathway

The insulin growth factor pathway (IGF), a complex one with multiple receptors and ligands, is involved in essential steps of cancer development such as survival, proliferation, and metastases.1 Although expression of IGF signaling seems to have prognostic significance, predictive factors for benefit have not yet been identified. Cross talk between the IGF and other pathways, particularly EFGR, may be important for understanding and mitigating resistance. Several compounds, including monoclonal antibodies and tyrosine kinase inhibitors, are currently under clinical investigation for inhibition of the IGF pathway.

Diagnostic and prognostic role of the insulin growth factor pathway members insulin-like growth factor-II and insulin-like growth factor binding protein-3 in serous effusions

We recently reported on higher expression of the insulin-like growth factor pathway genes IGF-II and IGFBP3 in serous ovarian/peritoneal carcinoma compared to malignant peritoneal mesothelioma. The present study analyzed the diagnostic and clinical role of these proteins in serous effusions. Effusions (n = 327), including 294 carcinomas (205 ovarian, 48 breast, 17 cervical/endometrial, 12 lung, 12 gastrointestinal/genitourinary) and 33 malignant mesotheliomas, were immunostained for insulin-like growth factor-II and insulin-like growth factor binding protein-3. Surgical ovarian carcinoma (n = 124) and peritoneal mesothelioma (n = 18) specimens were additionally studied. Insulin-like growth factor binding protein-3 levels were measured in 148 effusion supernatants (114 ovarian carcinomas, 18 breast carcinomas, 16 mesotheliomas) using enzyme-linked immunosorbent assay. Insulin-like growth factor binding protein-3 promoter methylation was analyzed in 11 ovarian carcinoma effusions. Insulin-like growth factor binding protein-3 (P = .002) and insulin-like growth factor-II (P < .001) expression by immunohistochemistry was significantly higher in carcinomas compared to mesotheliomas, with diagnostic sensitivity of 77% and 70% and specificity of 55% and 70%, respectively. In surgical specimens, insulin-like growth factor binding protein-3 expression was higher in ovarian carcinomas compared to peritoneal mesotheliomas (P = .007), whereas insulin-like growth factor-II expression was comparable (P = .505). Insulin-like growth factor binding protein-3 levels by enzyme-linked immunosorbent assay were comparable in the 3 analyzed cancer types. Insulin-like growth factor binding protein-3 promoter methylation was found in 6 of 11 effusions. High insulin-like growth factor binding protein-3 expression in prechemotherapy and high insulin-like growth factor-II expression in postchemotherapy ovarian carcinoma effusions correlated with poor overall survival (P = .031 and P = .024, respectively). Insulin-like growth factor-II expression in postchemotherapy effusions was an independent prognostic factor in Cox multivariate analysis (P = .04). In conclusion, insulin-like growth factor-II and insulin-like growth factor binding protein-3 are more frequently expressed in metastatic carcinomas compared to mesothelioma in effusions but are less specific than currently used markers. Insulin-like growth factor-II and insulin-like growth factor binding protein-3 may be novel prognostic markers in metastatic ovarian carcinoma.

Transformation of human urothelial cells (UROtsa) by as and cd induces the expression of keratin 6a.

BackgroundCadmium and arsenite can directly and malignantly transform the UROtsa cell line. The tumor heterotransplants produced from these transformed cells have histologic features consistent with human bladder cancer. Previous microarray analysis of total RNA from the parental and transformed cells suggested that keratin 6a was overexpressed as a result of cell transformation.ObjectivesOur goals were to verify overexpression of keratin 6a in Cd2+- and As3+-transformed UROtsa cells, the corresponding tumor heterotransplants, and human bladder cancer biopsy specimens and to assess what factors may be involved in keratin 6a overexpression.MethodsExpression was assessed with real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry. We used the effect of addition and deletion of potential growth factors in the cell culture growth medium to assess possible pathways used in keratin 6a overexpression.ResultsCd2+- and As3+-transformed cells grown in serum-containing growth medium, as well as the derived tumor heterotransplants, overexpressed keratin 6a mRNA and protein compared with UROtsa cells grown in serum-containing growth medium. Immunostaining of keratin 6a in tumor heterotransplants showed focal staining of the tumor cells that was localized to the cytoplasm. Focal immunostaining of keratin 6a was also found in some but not all archival patient specimens of high-grade bladder cancer, confirming translation of the results to human bladder cancer. Studies on growth factor deletion and addition indicated that the level of keratin 6a expression was regulated by the presence of both insulin and epidermal growth factor (EGF). In contrast, growth factors had no effect on the elevated levels of keratin 6a expression found in transformed UROtsa cells.ConclusionsOur present studies suggest that keratin 6a expression may be a biomarker for malignant urothelial cells that possess an activated EGF and or insulin growth factor pathway.