Depletion of insulin-like growth factor 1 receptor increases radiosensitivity in colorectal cancer

BackgroundAlthough radiation therapy for advanced colorectal cancer (CRC) is very effective in some patients, treatment resistance limits its efficacy. Insulin-like growth factor 1 receptor (IGF1R) can affect tumor responsiveness and sensitivity to radiation in several cancer types. Herein, we studied the underlying function of IGF1R in the resistance of advanced CRC to radiation therapy and the possible use of drugs targeting IGF1R to overcome this resistance in patients with CRC.MethodsDifferences in the expression levels of the IGF1R were assessed in CRC samples from patients who were radiosensitive or radioresistant. Two radio-resistant colorectal cancer cell lines, SW480 and HT29, were selected for in vitro studies, and the involvement of the IGF1R in their radiation resistance was elucidated by suppressing its expression through a targeted siRNA and through the use of a specific IGF1R inhibitor, BMS-754807. We assessed radiosensitivity in these human CRC cells lines by examining their proliferation and colony formation, as well as cell cycle analysis. Activation of the Akt pathway was assessed using western blotting.ResultsCompared with tissues from radiosensitive patients, higher IGF1R expression levels were found in patients with radiation-resistant colorectal cancer, while BMS-754807 had improved radiosensitivity and reversed radiation tolerance in both colorectal cancer cell lines. Pre-treatment with BMS-754807 prior to irradiation inhibited Akt phosphorylation, induced cell cycle arrest, and increased DNA damage. Therefore, the IGF1R contributes to radiation resistance of CRC cells in vitro.ConclusionsThis study supports the notion that the radiosensitivity of radiation-resistant colorectal cancer cells can be enhanced by directly targeting IGF1R expression or activity. Ultimately, the combination of radiotherapy with IGF1R targeted inhibitors could potentially increase its effectiveness in the treatment of advanced colorectal cancer.     

Recombinant human stem cell factor does exert minor stimulation of growth in small cell lung cancer and melanoma cell lines

We have previously reported on the stimulation of clonal growth of a glioblastoma cell line by rhSCF (Berdel et al., Cancer Res 1992, 52, 3498–3502). Within an extensive screening programme of haematopoietic growth factor activity on malignant cells, the effects of rhSCF were further tested on the growth of 29 different human cell lines derived from a wide range of solid tumours, among them six lung cancers and five melanomas. RhSCF (0,1,10, 100 ng/ml) was tested in a human tumour cloning assay (HTCA) which reliably detects growth modulation of tumour cells by cytokines. Additionally, a tritiated thymidine uptake test was used. Growth of 27 of the 29 cell lines tested was not affected by rhSCF. However, growth of the small cell lung cancer (SCLC) cell line HTB 120 was slightly stimulated (1.5 fold that of controls), and that of the melanoma cell line Me Wowas stimulated up to 1.3-fold. This activity was eliminated dose-dependently by the tyrosine kinase inhibitor, genistein. We further analysed the cell lines for expression of the proto-oncogene C-KIT and its ligand SCF. All melanoma and lung cancer cell lines expressed SCF as assessed at the mRNA level. Northern blotting also revealed clear C-KIT mRNA expression in three melanoma (HAS, MeWo, SK-MEL-28), one NSCLC (HTB 53), and four SCLC cell lines (HTB 119, HTB 120, HTB 171, HTB 175). Furthermore, C-KIT protein expression was detected by flow cytometric analysis on the cell surface of MeWo, HTB 119 and HTB 120 cells. Our data indicate that SCF can be operative in growth modulation of non-haematopoietic malignant cells, especially SCLC and melanoma. However, our extensive screening of SCF/tumour cell interaction shows that this interaction is rare and makes potential hazards, such as tumour stimulation upon clinical use of rhSCF in conjunction with chemotherapy in cancer patients, unlikely for the majority of other tumour histologies.     

Dual inhibition of epidermal growth factor and insulin-like 1 growth factor receptors reduce intestinal adenoma burden in the Apc(min/+) mouse

Background:

Identification of early molecular pathway changes may be useful as biomarkers for tumour response/resistance prediction, and here we provide direct in vivo proof of this concept. The type 1 insulin-like growth factor receptor (IGF1R) has been implicated in various aspects of adenoma development and metastasis. We show here that, in murine intestinal adenomas acutely exposed to a small molecular inhibitor of EGFR (gefitinib), there is concurrent suppression of EGFR downstream signalling and induction of IGF signalling. We therefore tested the hypothesis that blockade of EGFR signalling was being tempered by compensatory activation of the IGF pathway by examining the effect of chronic suppression of IGF1R using AZ12253801, a small molecular tyrosine kinase inhibitor of IGF1R.

Methods:

Male Apc^min/+ mice with an intestinal tumour burden were exposed to a single dose of an inhibitor against EGFR (gefitinib), IGF1R (AZ12253801), 0.5% Tween 80 or combined EGFR/IGF1R inhibitor and culled 4 h post dosing. Tumour tissue was analysed to detect the early molecular pathways induced and anti-tumour phenotypic changes. Cohorts of male Apc^min/+ mice (n=15–17) were subsequently treated with gefitinib for a period of 8 weeks and subsequently exposed to single (either gefitinib or AZ12253801) or combined (gefitinib and AZ12253801) therapy. We also included a vehicle-treated cohort, which was never exposed to gefitinib and became symptomatic of the disease by day 150.

Results:

Both single treatments delayed the onset of disease symptoms. Combined dosing with gefitinib and AZ12253801 similarly delayed the onset of symptoms, and at 200 days suppressed small intestinal tumourigenesis more effectively than either treatment alone (median small intestinal adenoma volume (47 mm³ (comb) vs 248 mm³ (AZ12253801), P=0.0003 and 47 mm³ (comb) vs 123 mm³ (gefitinib), P=0.0042, Mann–Whitney (two-sided) test).

Conclusion:

Our data provide evidence in support of the use of combinatorial therapy, and establishes the need to further define the precise benefit in vivo.     

A humanised anti-IGF-1R monoclonal antibody (AVE1642) enhances Bortezomib-induced apoptosis in myeloma cells lacking CD45

The humanised form of an antagonistic anti-IGF-1R mAb (AVE1642) selectively inhibits the growth of CD45^neg myeloma cells. AVE1642 strongly increased bortezomib-induced apoptosis, correlated with an increase of Noxa expression. These results support the therapeutic use of anti-IGF-1R/bortezomib in CD45^neg Myeloma patients, particularly those with the most aggressive form, t(4,14).     

Oncogenic functions of IGF1R and INSR in prostate cancer include enhanced tumor growth,cell migration and angiogenesis

We scrutinized the effect of insulin receptor (INSR) in addition to IGF1R in PCa using in vitro and in vivo models. In-vitro overexpression of IGF1R and INSRA, but not INSRB increased cell proliferation, colony formation, migration, invasion and resistance to apoptosis in prostate cancer cells (DU145, LNCaP, PC3). Opposite effects were induced by downregulation of IGF1R and total INSR, but not INSRB. In contrast to tumor cells, non-cancerous epithelial cells of the prostate (EP156T, RWPE-1) were inhibited on overexpression and stimulated by knockdown of receptors. In-vivo analyses using the chicken allantoic membrane assay confirmed the tumorigenic effects of IGF1R and INSR. Apart from promoting tumor growth, IGF1R and INSR overexpression also enhanced angiogenesis indicated by higher vessel density and increased number of desmin-immunoreactive pericytes. Our study underscores the oncogenic impact of IGF1R including significant effects on tumor growth, cell migration, sensitivity to apoptotic/chemotherapeutic agents and angiogenesis, and characterizes the INSR, in particular the isoform INSRA, as additional cancer-promoting receptor in prostate cancer. Both, the insulin-like growth factor receptor 1 and the insulin receptor exert oncogenic functions, thus proposing that both receptors need to be considered in therapeutic settings.     

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.     

Abcc10 status affects mammary tumour growth,metastasis, and docetaxel treatment response

Background:

Resistance to chemotherapeutic agents is a major obstacle to cancer treatment. A group of ABC efflux pumps, the Multidrug Resistance Proteins, is a source of resistance. Herein, we investigated the role of ABCC10 in mammary tumours, given the important role we have defined for ABCC10 in transporting taxanes, and the recognition that some ABCC proteins have roles in tumour growth.

Methods:

ABCC10 expression was correlated to human breast cancer subtype using breast tissue microarrays. Real-time quantitative PCR and western blot analysis were used to examine ABCC10 expression in human breast cancer lines. Abcc10^−/− mice were crossed to MMTV-PyVmT mice to produce Abcc10^−/− vs Abcc10^+/+ mammary tumours and derivative cell lines. We used allograft and cellular assays to perform baseline and drug sensitization analysis of tumours and cell lines.

Results:

Clinical sample analyses indicated that ABCC10 was more highly expressed in Her2+ and ER+ than in Her2−, ER−, and triple-negative breast cancer. Unexpectedly, PyVmT; Abcc10^−/− tumours grew more rapidly than PyVmT; Abcc10^+/+ tumours and were associated with significantly reduced apoptosis and metastasis. PyVmT; Abcc10^−/− lines were less migratory than PyVmT; Abcc10^+/+ lines. Finally, we showed increased survival of docetaxel-treated MMTV-PyVmT; Abcc10^−/− mice compared with wild-type mice.

Conclusions:

These data identify roles for Abcc10 in breast cancer pathogenesis and in vivo docetaxel resistance.     

Insulin-like growth factor (IGF)-binding protein-4 inhibits colony formation of colorectal cancer cells by IGF-independent mechanisms

Effects of insulin-like growth factor-binding protein-4 (IGFBP-4) on proliferation, colony formation, and cell migration were assessed in IGF-sensitive and -insensitive colorectal cancer cell lines. In IGF-insensitive Isreco-1 cells, overexpression of IGFBP-4 reduced colony formation but not cell proliferation and migration, whereas exogenous IGF-II had no effect. In IGF-dependent LS1034 cells, IGFBP-4 inhibited all parameters of growth tested, whereas IGF-II partially restored reduced proliferation and cell migration only. In Isreco-2 cells, which lack endogenous IGF expression but are IGF sensitive, colony formation was also reduced by IGFBP-4. Therefore, specific parameters of malignant progression of colon carcinoma cells are distinctly affected by IGF-dependent and IGF-independent effects of IGFBP-4.     

hERG1 channels drive tumour malignancy and may serve as prognostic factor in pancreatic ductal adenocarcinoma

Background:

hERG1 channels are aberrantly expressed in human cancers. The expression, functional role and clinical significance of hERG1 channels in pancreatic ductal adenocarcinoma (PDAC) is lacking.

Methods:

hERG1 expression was tested in PDAC primary samples assembled as tissue microarray by immunohistochemistry using an anti-hERG1 monoclonal antibody (α-hERG1-MoAb). The functional role of hERG1 was studied in PDAC cell lines and primary cultures. ERG1 expression during PDAC progression was studied in Pdx-1-Cre,LSL-Kras^G12D/+,LSL-Trp53^R175H/+ transgenic (KPC) mice. ERG1 expression in vivo was determined by optical imaging using Alexa-680-labelled α-hERG1-MoAb.

Results:

(i) hERG1 was expressed at high levels in 59% of primary PDAC; (ii) hERG1 blockade decreased PDAC cell growth and migration; (iii) hERG1 was physically and functionally linked to the Epidermal Growth Factor-Receptor pathway; (iv) in transgenic mice, ERG1 was expressed in PanIN lesions, reaching high expression levels in PDAC; (v) PDAC patients whose primary tumour showed high hERG1 expression had a worse prognosis; (vi) the α-hERG1-MoAb could detect PDAC in vivo.

Conclusions:

hERG1 regulates PDAC malignancy and its expression, once validated in a larger cohort also comprising of late-stage, non-surgically resected cases, may be exploited for diagnostic and prognostic purposes in PDAC either ex vivo or in vivo.     

Stem cell factor (SCF) synergizes with megakaryocyte colony stimulating activity in post-irradiated aplastic plasma in stimulating human megakaryocytopoiesis

Plasma obtained from lethally irradiated animals contains a megakaryocyte (MK) growth factor which has recently been identified as the ligand for the c-mpl receptor and has been named thrombopoietin (TPO). We demonstrate that post-irradiation aplastic canine plasma (PICS-J) and plasma from a human subject (ML) who was accidentally exposed to lethal irradiation, contain high levels of this activity, which support both MK proliferation and maturation in a dose-dependent manner. These plasma were far more active in stimulating human MK colony formation than other types of thrombocytopenic plasma or a number of exogenously added human recombinant cytokines and their combinations. The addition of stem cell factor (SCF), which alone has a minimal stimulatory affect, to post lethal-irradiation plasma provided a synergistic stimulation of megakaryocytopoiesis both in colony assays and liquid cultures. In colony assays, the combination of SCF with PICS-J or ML almost doubled the number of burst forming units (BFU-MK) and provided a 1.5-fold increase in colony forming units (CFU-MK). A 1.6-fold increase in the number of CD34⁺ BM cell-derived MK colonies was also elicited. In liquid cultures, the presence of both SCF and PICS-J or ML induced the appearance of a high proportion of CD34⁺ (6.56% vs 0.6% control) and CD41⁺ (3.5% vs 1.2% control) cells after 3 days in culture. By day 10, 66.8 xl0⁴ CD41⁺ cells and 29.8xl0⁴ CD34⁺ cells were derived from 2x10⁶ BMMC originally seeded. We propose that these unique plasma, which do not contain elevated levels of IL-6, IL-3, GM-CSF, IL-1β, erythropoietin or SCF, probably contain high levels of TPO. The addition of SCF to the post-irradiation plasma provides a synergistic stimulation of megakaryocytopoiesis which may become relevant for future clinical application.     

Silencing NOTCH signaling causes growth arrest in both breast cancer stem cells and breast cancer cells

Background:

Breast cancer stem cells (BCSCs) are characterized by high aldehyde dehydrogenase (ALDH) enzyme activity and are refractory to current treatment modalities, show a higher risk for metastasis, and influence the epithelial to mesenchymal transition (EMT), leading to a shorter time to recurrence and death. In this study, we focused on examination of the mechanism of action of a small herbal molecule, psoralidin (Pso) that has been shown to effectively suppress the growth of BSCSs and breast cancer cells (BCCs), in breast cancer (BC) models.

Methods:

ALDH⁻ and ALDH⁺ BCCs were isolated from MDA-MB-231 cells, and the anticancer effects of Pso were measured using cell viability, apoptosis, colony formation, invasion, migration, mammosphere formation, immunofluorescence, and western blot analysis.

Results:

Psoralidin significantly downregulated NOTCH1 signaling, and this downregulation resulted in growth inhibition and induction of apoptosis in both ALDH⁻ and ALDH⁺ cells. Molecularly, Pso inhibited NOTCH1 signaling, which facilitated inhibition of EMT markers (β-catenin and vimentin) and upregulated E-cadherin expression, resulting in reduced migration and invasion of both ALDH⁻ and ALDH⁺ cells.

Conclusion:

Together, our results suggest that inhibition of NOTCH1 by Pso resulted in growth arrest and inhibition of EMT in BCSCs and BCCs. Psoralidin appears to be a novel agent that targets both BCSCs and BCCs.     

Evaluation of IGF1R and phosphorylated IGF1R as targets in HER2-positive breast cancer cell lines and tumours

Insulin-like growth factor-1 receptor (IGF1R) signalling is implicated in resistance to trastuzumab. However, the benefit of co-targeting HER2 and IGF1R has not been extensively studied, and the relationship between activated IGF1R and clinical response to trastuzumab has not been reported. This study aimed to evaluate the combination of trastuzumab with IGF1R tyrosine kinase inhibitors (TKIs) in a panel of HER2-positive breast cancer cell lines, and to examine the relationship between IGF1R expression and activation and response to trastuzumab in HER2-positive breast cancer patients. The anti-proliferative effects of trastuzumab combined with IGF1R TKIs BMS-536924 or NVP-AEW541 were measured in nine HER2-positive cell lines. IGF1R and phosphorylated IGF1R/insulin receptor (pIGF1R/IR) were measured by immunohistochemistry in 160 tumour samples from trastuzumab-treated patients (ICORG 06-22). The HER2-positive cell lines displayed varying sensitivity to IGF1R TKIs alone (IC₅₀s: 0.7 to >10???M). However, when combined with trastuzumab, a significantly enhanced effect was observed in five cell lines treated with BMS-536924, and three with NVP-AEW541. While IGF1R levels correlated with reduced response to NVP-AEW541 alone, neither IGF1R nor pIGF1R were predictive of response to BMS-536924 or NVP-AEW541 in combination with trastuzumab. Low HER2 levels correlated with response to BMS-536924 in combination with trastuzumab. Akt levels correlated with improved response to trastuzumab and NVP-AEW541 (P?=?0.039). Cytoplasmic IGF1R staining was observed in all tumours, membrane IGF1R was detected in 13.8?%, and pIGF1R/IR was detected in 48.8?%. Although membrane IGF1R staining was associated with larger tumour size (P?=?0.041), and lower tumour grade (P?=?0.024), no association between IGF1R or pIGF1R/IR and patient survival was observed. In conclusion, while neither IGF1R expression nor activation was predictive of response to trastuzumab, these pre-clinical data provide evidence that co-targeting HER2 and IGF1R may be beneficial in some HER2-amplified breast cancers.     

miR-133a suppresses ovarian cancer cell proliferation by directly targeting insulin-like growth factor 1 receptor

The microRNA miR-133a is dysregulated in many types of cancer, but the underlying mechanism remains largely unknown. In this study, we showed that the expression level of miR-133a was reduced in ovarian cancer tissues compared with normal ovaries. Ectopic expression of miR-133a significantly inhibited ovarian cancer cell proliferation and colony formation, and induced G1-phase cell cycle arrest, whereas decreased miR-133a expression dramatically enhanced cell proliferation and colony formation. Importantly, miR-133a overexpression suppressed in vivo tumor growth in nude mice models. Through in silico search, we found that the 3′-untranslated region (UTR) of insulin-like growth factor 1 receptor (IGF1R) contains an evolutionarily conserved miR-133a binding site. miR-133a overexpression repressed IGF1R-3′UTR reporter activity, and reduced the mRNA and protein levels of endogenous IGF1R. Rescue experiments showed that ectopic expression of IGF1R significantly promoted the proliferation of ovarian cancer cells stably overexpressing miR-133a. Taken together, these findings indicate that miR-133a is an important regulator in ovarian cancer, and that its suppressive effects are mediated by targeting IGF1R.     

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation

Background:

Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development.

Methods:

To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53^lox/lox/FAK^+/+/WapCre, p53^lox/lox/FAK^flox/+/WapCre and p53^lox/lox/FAK^flox/−/WapCre mice, and mice with WapCre-mediated conditional expression of p53^R270H, the mouse equivalent of human p53^R273H hot spot mutation, together with conditional deletion of FAK, P53^R270H/+/FAK^lox/+/WapCre and p53^R270H/+/FAK^flox/−/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours.

Results:

Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53^R270H-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures.

Conclusions:

Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53^R270H mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53^R270H-induced mammary tumour development.     

RUNX1 haploinsufficiency results in granulocyte colony-stimulating factor hypersensitivity

RUNX1/AML1 is among the most commonly mutated genes in human leukemia. Haploinsufficiency of RUNX1 causes familial platelet disorder with predisposition to myeloid malignancies (FPD/MM). However, the molecular mechanism of FPD/MM remains unknown. Here we show that murine Runx1^+/− hematopoietic cells are hypersensitive to granulocyte colony-stimulating factor (G-CSF), leading to enhanced expansion and mobilization of stem/progenitor cells and myeloid differentiation block. Upon G-CSF stimulation, Runx1^+/− cells exhibited a more pronounced phosphorylation of STAT3 as compared with Runx1^+/+ cells, which may be due to reduced expression of Pias3, a key negative regulator of STAT3 signaling, and reduced physical sequestration of STAT3 by RUNX1. Most importantly, blood cells from a FPD patient with RUNX1 mutation exhibited similar G-CSF hypersensitivity. Taken together, Runx1 haploinsufficiency appears to predispose FPD patients to MM by expanding the pool of stem/progenitor cells and blocking myeloid differentiation in response to G-CSF.     

Dexamethasone-induced cell death is restricted to specific molecular subgroups of multiple myeloma

Due to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic heterogeneity of MM because sensitive cell lines were restricted to MAF and MMSET signature subgroups, whereas all CCND1 cell lines (n = 10) were resistant to dexamethasone. We demonstrated that the glucocorticoid receptor expression was an important limiting factor for dexamethasone-induced cell death and we found a correlation between glucocorticoid receptor levels and the induction of glucocorticoid-induced leucine zipper (GILZ) under dexamethasone treatment. By silencing GILZ, we next demonstrated that GILZ is necessary for Dex induced apoptosis while triggering an imbalance between anti- and pro-apoptotic Bcl-2 proteins. Finally, the heterogeneity of the dexamethasone response was further confirmed in vivo using myeloma xenograft models. Our findings suggested that the effect of dexamethasone should be re-evaluated within molecular subgroups of myeloma patients to improve its efficacy and reduce its adverse effects.