Regulation of protumorigenic pathways by Insulin like growth factor binding protein2 and its association along with β-catenin in breast cancer lymph node metastasis

The Insulin like Growth Factor binding proteins (IGFBP) are a family of six proteins that bind with high affinity to Insulin like growth factors (IGF-I and IGF-II), prolong their half-life in circulation and thereby regulate IGF actions. Insulin like growth factor binding protein 2 (IGFBP2) is the second most abundant IGFBP in circulation and in a context dependent manner it can either inhibit or potentiate the actions of IGF [1], thereby modulating the prosurvival and/or mitogenic effects of IGF. Elevated expression of IGFBP2 has been observed in multiple malignancies, including Glioblastoma multiforme [2‐4], ovarian [5, 6], pancreatic [7], gastric [8], prostate [9], colon [10], breast [11, 12], leukemia [13] and thyroid cancer [14]. In addition, increased expression of IGFBP2 has been correlated with poor prognosis in prostate, glioblastoma and colon cancers [15‐18]. It has been reported that IGFBP2 inhibits the IGF dependent proliferation of normal cells while in tumor cells, it promotes proliferation in an IGF1R dependent or independent manner [19, 20]. Pro proliferative action of IGFBP2 has been reported in prostate, ovarian and colon cancer cells and non-transformed rat osteoblasts [19, 21‐24]. IGFBP2 expression has also been shown to enhance migration and invasion in glioma, ovarian and bladder cancer cells [3, 25‐27]. Recent studies in glioma implicate IGFBP2 in the activation of PI3K Akt pathway [28], integrin/ILK/NF-B network which drives glioma progression in mice [29] and binding to integrin α5 [30] that brings about increased migration and invasion. In breast cancer, IGFBP2 over expression has been shown to confer drug resistance [11] and increased expression has been reported to correlate with lymph node metastasis In T1 breast carcinomas [31]. However, mechanisms that govern IGFBP2 actions in breast cancers are poorly understood.

In the present study, to elucidate the cellular pathways influenced by IGFBP2 in breast cancer, gene expression profiling of IGFBP2 knockdown breast cancer cells was compared with the expression profile of IGFBP2 positive breast tumors. Our results highlight regulation of cell cycle and Wnt signaling pathways by IGFBP2. Most significantly, our data shows for the first time that the concomitant over expression of IGFBP2 and β-catenin in breast cancer is associated with increased incidence of lymph node metastasis.

Enhanced expression of IGFBP2 is associated with a large number of malignant cancers that include tumors of breast, ovarian, glioma and prostate. Primarily known for its growth inhibitory actions in physiological context, IGFBP2 has now been shown to promote growth and tumorigenesis in numerous cancer cells such as glioma, prostate and colon cancers [15‐18]. To gain further insights into the role of IGFBP2 in breast cancer, we have attempted to identify the molecular players in IGFBP2 associated tumorigenesis in breast cancer. To elucidate the molecular targets of IGFBP2, we perturbed IGFBP2 expression by shRNA and the differential gene expression was determined using whole genome microarrays. IGFBP2 knockdown resulted in significant changes in the expression of genes associated with cellular proliferation and tumorigenicity. The down regulated genes were found to be associated with several pathways, notably Cell cycle, p53 and Wnt pathways as revealed by GSEA. Comparison of our data with a previous microarray study of IGFBP2 regulated genes in glioma cells [29] revealed an overlap of about 22% genes with wild type IGFBP2 over expressing cells and 23% genes with RGE mutant IGFBP2 over expressing cells. Pathway comparisons revealed Cell cycle, p53 signaling, oxidative phosphorylation, nucleotide metabolism and Wnt signaling pathway to be common among the two data sets (Additional file 8: Figure S4). To further validate these results in breast cancer tissues, we performed whole genome expression analysis in 19 breast tumors which were categorized as IGFBP2 positive or negative based on immunohistochemical staining pattern. Compared to IGFBP2 negative tumors, IGFBP2 positive tumors showed increased expression of genes belonging to MAPK signaling, Focal adhesion and Wnt signaling.

IGFBP2 correlation with proliferation has been studied extensively in several tumor cells including in breast cancer cells. The effect of IGFBP2 on proliferation has been shown to be context dependent. In prostate, ovarian, nephroblastoma cells, it has a pro proliferative action [19, 21‐24]. In contrast IGFBP2 has an antiproliferative effect on HEK, Hs578T [32, 33]. Our data on the regulation of different pathways such as MAPK, Cell cycle, Focal adhesion and Wnt corroborate the reported functional significance of IGFBP2 with respect to its pro proliferative and tumor promoting roles in breast cancer cells.

One of the important and novel findings from this study is the regulation of Wnt signaling pathway genes by IGFBP2. So far, only IGFBP4 has been reported to activate Wnt signaling pathway in renal cell carcinoma [34]. Activation of canonical Wnt signaling promotes tumorigenesis by regulating cell survival, proliferation and invasion of many cancers [35]. In numerous tumors cytoplasmic and/or nuclear accumulation of β-catenin has been shown to be a strong indicator of aberrant Wnt pathway activation. Elevated cytosolic and nuclear accumulation of β-catenin has been associated with a variety of malignancies and inversely correlated with patient survival [36‐39], Wnt activation leads to stabilization and translocation of β-catenin from cytoplasm to the nucleus where it associates with T-cell factor (TCF)/lymphocyte enhancer transcription (LEF) factors to activate target genes that are involved in cell survival, proliferation, and invasion [40, 41]. In order to establish Wnt pathway activation by IGFBP2, we examined the canonical Wnt signaling target, β-catenin in IGFBP2 knockdown breast cancer cells. Compared to Vector transfected cells, IGFBP2 knockdown cells showed remarkably decreased levels of β-catenin. When IGFBP2 was re expressed in the knockdown cells, as expected there was substantial increase in β-catenin levels indicating that IGFBP2 regulates β-catenin. Interestingly, inhibition of IGF1R or integrin signaling resulted in the loss of β-catenin regulation by IGFBP2. These data suggest that IGFBP2 acts through IGF1R and integrin pathways in the regulation of β-catenin. Although the mechanisms are not clear, recently Uzoh et al. demonstrated an increased proliferation of prostate cancer cells by IGFBP2 in an IGF1R dependent manner [20]. It is also known that IGF independent actions of IGFBP2 are mediated by the activation of integrin signaling through RGD motif present in the C-terminal region of IGFBP2 protein [30]. Role of integrin receptors in pro-tumorigenic action of tumor cells is well established [42, 43]. Hence, it is conceivable that activation of integrin signaling by IGFBP2 leading to FAK phosphorylation may be an important step in the activation of IGF1R by IGFBP2. In congruence with this, it has been reported that activated FAK phosphorylates and stabilizes IGF1R in mouse embryonic fibroblast [44]. Very recently, IGFBP2 in association with IGF1 was found to activate IGF1R in endothelial cells [45]. Taken together, regulation of Wnt pathway by IGFBP2 involves FAK and IGF1R in breast carcinogenesis. However, the mechanism (s) by which FAK and IGF1R signaling converge on the regulation of Wnt pathway by IGFBP2 needs further investigations.

Another important finding from our data is the correlation of IGFBP2 over expression with elevated β-catenin levels in breast tumors. In humans, breast tumors frequently exhibit elevated levels of IGFBP2 [12] and β-catenin, with higher expression levels of β-catenin correlating with a decreased patient survival [39]. In mice, over expression of an activated β-catenin leads to the development of mammary hyperplasia and adenocarcinomas [46]. These studies coupled with our data suggest that regulation of β-catenin could be an important step for the pro-tumorigenic actions of IGFBP2. Most significantly, when both IGFBP2 and β-catenin expression was correlated with the lymph node status of breast cancers, we found a significant association of IGFBP2 and β-catenin staining with increased lymph node metastasis in comparison with tumors which did not show staining for either protein. Interestingly, in a previous report, expression of IGFBP2 and IGFBP5 were correlated with increased lymph node metastasis in T1 breast carcinoma. However our data shows a significant positive correlation of IGFBP2 and β-catenin in lymph node metastasis. Hence, evaluation of IGFBP2, IGFBP5 along with β-catenin may provide a stronger predictive value for the prognosis of breast cancer.

This study highlights the pathways and genes regulated by IGFBP2 in breast cancer. Most importantly, this study reports regulation of β-catenin by IGFBP2 and their association in the lymph node metastasis. These findings are highly relevant in the prediction of breast cancer progression.

All the tissues for this study were collected after obtaining written informed consent from the patients. This study and the protocols were approved by the Institutional Ethics Committee of Kidwai Memorial Institute of Oncology, where the patients were treated.