IGF-1R siRNA Research Articles

Expression of IGF-1R in esophageal squamous cell carcinoma and the effect of its silencing by siRNA on the proliferation of esophageal cancer EC9706 cells in vitro

To explore the correlation of IGF-1R expression with clinical features of esophageal squamous cell carcinoma (ESCC) and to investigate the effect of silencing IGF-1R by siRNA on the proliferation of esophageal cancer cell line EC9706 cells. Immunohistochemistry was used to detect the expresion of IGF-1R in 80 specimens of ESCC and 18 specimens of normal esophageal mucosa. IGF-1R siRNA was transfected into esophageal squamous cell carcinoma EC9706 cells, and the effect of RNAi was assessed by Western blot. The proliferation of EC9706 cells was determined by drawing growth curve, MTT assay and plate colony-forming assay. The total and strong positive rates of IGF-1R expression were 86.3% and 51.3% in ESCC, and 61.1% and 11.1% in normal esophageal epithelium, respectively. The total and strong positive rates of IGF-1R expression in patients with lymph node metastasis were 94.4% and 74.1%, significantly higher than 69.2% and 3.9%, respectively, in those without lymph node metastasis (P<0.01). A significantly higher IGF-1R expression was associated with lower histological grade (P<0.05). The total and strong rates of IGF-1R expression in 39 patients of stages III and IV were 97.4% and 71.8% , significantly higher than the 75.6% and 31.7%, respectively, in 41 cases of stages I and II (P<0.01). IGF-1R RNAi significantly inhibited IGF-1R expression and the growth of EC9706 cells. The clone formation rate of RNAi-IGF-1R transfected cells was 19.1%, significantly lower than that of 52.3% in non-transfected cells and 49.0% in empty vector-transfected EC9706 cells (P<0.05). The overexpression of IGF-1R is colerated with lymph node metastasis, differentiation and clinical stage. Down-regulation of IGF-1R can inhibit the proliferation of esophageal cancer EC9706 cells in vitro.

Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways.

BackgroundObesity is a global phenomenon and is associated with various types of cancer, including colon cancer. There is a growing interest for safe and effective bioactive compounds that suppress the risk for obesity-promoted colon cancer. Resveratrol (trans-3, 4', 5,-trihydroxystilbene), a stilbenoid found in the skin of red grapes and peanuts suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms, however, resveratrol effects on obesity-promoted colon cancer are not clearly established.MethodsWe investigated the anti-proliferative effects of resveratrol on HT-29 and SW480 human colon cancer cells in the presence and absence of insulin like growth factor-1 (IGF-1; elevated during obesity) and elucidated the mechanisms of action using IGF-1R siRNA in HT-29 cells which represents advanced colon carcinogenesis.ResultsResveratrol (100-150 μM) exhibited anti-proliferative properties in HT-29 cells even after IGF-1 exposure by arresting G0/G1-S phase cell cycle progression through p27 stimulation and cyclin D1 suppression. Treatment with resveratrol suppressed IGF-1R protein levels and concurrently attenuated the downstream Akt/Wnt signaling pathways that play a critical role in cell proliferation. Targeted suppression of IGF-1R using IGF-1R siRNA also affected these signaling pathways in a similar manner. Resveratrol treatment induced apoptosis by activating tumor suppressor p53 protein, whereas IGF-1R siRNA treatment did not affect apoptosis. Our data suggests that resveratrol not only suppresses cell proliferation by inhibiting IGF-1R and its downstream signaling pathways similar to that of IGF-1R siRNA but also enhances apoptosis via activation of the p53 pathway.ConclusionsFor the first time, we report that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis even in the presence of IGF-1 via suppression of IGF-1R/Akt/Wnt signaling pathways and activation of p53, suggesting its potential role as a chemotherapeutic agent.

IGF‐1R Regulation of Colon Cancer Stem Cells

Despite recent advances in therapeutics, recurrence of colon cancer occurs in 50% of the cases, which could partly be due to the presence of self renewing cancer stem‐like cells (CSCs) which are resistant to chemotherapy. We have observed that chemosurviving colon cancer cells are enriched in CSCs as evidenced by increased number of CD44+ cells, associated with activated growth factor receptors especially IGF‐1R (4.5‐fold). Depletion of IGF‐1R by si‐RNA results in growth inhibition of chemosurviving cells accompanied by a reduction in CD44 suggesting that IGF‐1R might target CSCs. Indeed, we observed a significant inhibition of colonosphere formation, a CSC property, in IGF‐1R si‐RNA transfected cells in p53 wt HCT‐116 cells accompanied by decrease in CD44 and CD166 expression and induction in miRNA which post transcriptional regulate gene expression. We identified miRNA‐363, a non‐p53 regulated miRNA, to be highly overexpressed (18.9‐fold) following IGF‐1R inhibition. In addition, miR‐215, a p53 regulated miRNA, which is normally downregulated in colon cancer, was upregulated by 2‐fold following IGF‐1R inhibition resulting in induction of its downstream effector p21waf1/cip1 only in p53 wild type colon cancer cells. We conclude that Inhibition of IGF‐1R results in reduction of CSC phenotype in colorectal cancer cells which could partly be due to induction of miRNAs ‐363 and ‐215.

SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation

Purpose. Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. Material and methods. Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. Results. Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 ± 0.08. Conclusion. These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone.

Commentary on Validation of the type 1 insulin-like growth factor receptor as a therapeutic target in renal cancer: Yuen JS, Akkaya E, Wang Y, Takiguchi M, Peak S, Sullivan M, Protheroe AS, Macaulay VM, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Headington, Oxford, United Kingdom

Expression of the type 1 insulin-like growth factor receptor (IGF1R) confers adverse prognosis in clear cell renal cell cancer (CC-RCC). We recently showed that IGF1R expression is inhibited by the von Hippel-Lindau (VHL) tumor suppressor, and the IGF1R is up-regulated in CC-RCC, in which VHL is frequently inactivated. We tested the hypothesis that IGF1R up-regulation mediates resistance to cancer therapeutics, evaluating the effects of IGF1R depletion on sensitivity to cytotoxic drugs, which are ineffective in RCC, and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, analogues of which have clinical activity in this tumor. This study used CC-RCC cells harboring mutant VHL and isogenic cells expressing functional VHL. Cells were transfected with nonsilencing control small interfering RNA (siRNA), or with one of two different IGF1R siRNAs. The more potent siRNA was modified by 2′-O-methyl derivatization for in vivo administration. CC-RCC cells expressing mutant VHL and higher IGF1R were more chemoresistant than cells expressing functional VHL. IGF1R depletion induced apoptosis, blocked cell survival, and sensitized to 5-fluorouracil and etoposide. These effects were significantly greater in CC-RCC cells expressing mutant VHL, supporting the hypothesis that IGF1R up-regulation makes a major contribution to the chemoresistance associated with VHL loss. IGF1R depletion also enhanced sensitivity to mTOR inhibition, at least in part due to suppression of rapamycin-induced Akt activation. Administration of stabilized IGF1R siRNA was shown to sensitize CC-RCC xenografts to rapamycin in vivo. These data validate IGF1R as a therapeutic target in CC-RCC, and support the evaluation of IGF1R-inhibitory drugs in patients with renal cancer.

Validation of the type 1 insulin-like growth factor receptor as a therapeutic target in renal cancer

Expression of the type 1 insulin-like growth factor receptor (IGF1R) confers adverse prognosis in clear cell renal cell cancer (CC-RCC). We recently showed that IGF1R expression is inhibited by the von Hippel-Lindau (VHL) tumor suppressor, and the IGF1R is up-regulated in CC-RCC, in which VHL is frequently inactivated. We tested the hypothesis that IGF1R up-regulation mediates resistance to cancer therapeutics, evaluating the effects of IGF1R depletion on sensitivity to cytotoxic drugs, which are ineffective in RCC, and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, analogues of which have clinical activity in this tumor. This study used CC-RCC cells harboring mutant VHL, and isogenic cells expressing functional VHL. Cells were transfected with nonsilencing control small interfering RNA (siRNA), or with one of two different IGF1R siRNAs. The more potent siRNA was modified by 2'-O-methyl derivatization for in vivo administration. CC-RCC cells expressing mutant VHL and higher IGF1R were more chemoresistant than cells expressing functional VHL. IGF1R depletion induced apoptosis, blocked cell survival, and sensitized to 5-fluorouracil and etoposide. These effects were significantly greater in CC-RCC cells expressing mutant VHL, supporting the hypothesis that IGF1R up-regulation makes a major contribution to the chemoresistance associated with VHL loss. IGF1R depletion also enhanced sensitivity to mTOR inhibition, at least in part due to suppression of rapamycin-induced Akt activation. Administration of stabilized IGF1R siRNA was shown to sensitize CC-RCC xenografts to rapamycin in vivo. These data validate IGF1R as a therapeutic target in CC-RCC, and support the evaluation of IGF1R-inhibitory drugs in patients with renal cancer.

Resveratrol Phenocopies the Suppressive Effects of Insulin‐like Growth Factor Receptor‐1 siRNA on IGF‐1 Promoted Colon Cancer Cell Proliferation

The insulin‐like growth factor (IGF) system is believed to mediate obesity‐related increase in the risk of colon cancer. IGFs activate the β‐catenin pathway, which plays a central role in the growth of colon cancer cells. Dietary resveratrol exerts inhibitory effects on cancers; however, its molecular mechanisms are not fully elucidated, particularly in relation to obesity‐promoted cancers. We hypothesized that resveratrol suppresses colon cancer cell proliferation by inhibiting activation of IGF receptor‐1 (IGF‐1R) in response to IGFs (elevated during obesity). IGF‐1 (10 nM) promoted proliferation of colon cancer cells, in vitro, as expected. Resveratrol (50–150 μM) or IGF‐1R siRNA (100 nM) inhibited (p &lt; 0.05) proliferation of colon cancer cells in response to both exogenous and autocrine IGFs. The relative levels of IGF‐1R were reduced (p &lt; 0.05) in cells treated with either resveratrol or IGF‐1R siRNA, in association with a loss (p &lt; 0.05) in Akt activation, GSK3β phosphorylation, and cyclin D1 levels. These results suggest that resveratrol effectively phenocopies the inhibitory properties of IGF‐1R siRNA and may thus be a potent chemopreventive/therapeutic agent against obesity‐associated increase in colon cancer risk. We will examine this possibility in future studies using in vivo models. Funded by TAES and AICR 058094.

SiRNA mediated the type 1 insulin-like growth factor receptor and epidermal growth factor receptor silencing induces chemosensitization of liver cancer cells

This study was to investigate if downregulation of IGF1R and EGFR by RNA interference (RNAi) would sensitize human liver cancer cells (HEPG2, Huh7 ) to adriamycin. HEPG2, Huh7 cell lines were transfected IGF1R siRNAs and EGFR siRNAs and IGF1R or EGFR mRNA level was determined by RT-PCR and Western-blot analysis. We investigated the effects of the adriamycin-induced apoptosis of these cells by TUNEL assay. Also we analyze caspase3, 8 and the phosphorylation levels of Akt and Erk by Western-blot. The p53 effect of adriamycin-induced cell death by inhibitors of EGFR/IGF1R is investigated by cell growth curves. Transfection of an IGF1R and EGFR siRNAs resulted in substantial loss of IGF1R and EGFR mRNA of HEPG2, Huh7 cells relative to the control case. EGFR siRNA and IGF1R siRNA treatments increased the adriamycin-induced apoptosis of these cells. IGF1R siRNA and EGFR siRNA enhance a caspase-dependent cell death program. The phosphorylation levels of Akt and Erk were reduced by the combination of the two agents. The facilitation of adriamycin-induced cell death by inhibitors of EGFR/IGF1R is p53-independent. The results indicate that the siRNA for IGF1R has a great potential for cancer therapy when combined with either a chemotherapeutic agent or siRNAs that targets EGFR.

Down-regulation of Type I Insulin-like Growth Factor Receptor Increases Sensitivity of Breast Cancer Cells to Insulin

The type I insulin-like growth factor receptor (IGF1R) and insulin receptor (IR) are structurally and functionally related heterotetrameric receptors. Activation of IGF1R has been shown to regulate breast cancer cell biology, and it has become an attractive therapeutic target. Most strategies have focused on targeting IGF1R alone without affecting IR levels given the known physiologic functions of IR. Human breast cancer cell lines and tissues revealed mRNA expression of both IGF1R and IR. Because alphabeta chains of IGF1R and IR form hybrid receptors, we hypothesized that agents solely targeting IGF1R may affect tumor biology mediated by IGF1R/IR hybrids and IR. We used small interfering RNA (siRNA) technology to specifically down-regulate IGF1R by 60% to 80% in the MDA-435/LCC6 cell line, which was sufficient to diminish activation of IGF1R by IGF-I. IGF1R down-regulation by siRNA did not affect IR levels but, interestingly, sensitized cells to insulin activation of downstream signaling pathways in several breast cancer cell lines. IGF1R siRNA treatment diminished hybrid receptor formation, suggesting that specific down-regulation of IGF1R resulted in enhanced holo-IR formation. In addition, IGF1R down-regulation increased insulin binding consistent with the formation of an increased number of holo-IR on the cell surface. Accordingly, insulin-stimulated glucose uptake was enhanced on IGF1R down-regulation. In conclusion, our data suggest that specific siRNA targeting of IGF1R alone in breast cancer increases insulin sensitivity. Because IR also activates signaling pathways similar to IGF1R in breast cancer cells, agents targeting both receptors may be necessary to disrupt the malignant phenotype regulated by this growth factor system.