Craig I. Campbell

IGF-II induces CREB phosphorylation and cell survival in human lung cancer cells

We have previously shown that lung tumors arising in MMTV-IGF-II transgenic mice displayed elevated levels of phosphorylated cAMP response element binding protein (CREB). To investigate the role that insulin-like growth factor II (IGF-II) and CREB play in human lung tumorigenesis, A549 and NCI-H358 cells were examined. In these cell lines, IGF-II administration enhances human tumor cell survival and CREB phosphorylation. Further, the effects of IGF-II on cell survival and CREB phosphorylation appeared to be mediated, at least in part, by activation of the Erk pathways, as inhibition of these signaling pathways reduced tumor cell survival and CREB phosphorylation. Specifically, Erk5 appeared as the predominant mediator of CREB phosporylation. To further verify the importance of CREB in human lung tumorigenesis, A549 and NCI-H358 cells were stably transfected with a vector containing a dominant negative CREB construct (KCREB). KCREB transfection significantly inhibited the soft agar growth of both human tumor cell lines. In contrast, overexpression of wild-type CREB in the normal human bronchial epithelial cell line, HBE135, enhanced soft agar growth. Therefore, our results indicate that CREB and its associated proteins play a significant role in lung adenocarcinoma and IGF-II induces CREB phosphorylation, at least in part, via the Erk5 signaling pathway.

Reversibility and recurrence of IGF-IR-induced mammary tumors

The type-I insulin-like growth factor receptor (IGF-IR) is frequently overexpressed in breast cancer and therapeutic agents targeting IGF-IR are currently in development. The ultimate success of anti-IGF-IR therapies will depend on the extent to which established tumors remain dependent upon IGF-IR signaling for sustained growth. To investigate the potential benefits and pitfalls of targeting IGF-IR, we used a doxycycline inducible mouse model of IGF-IR initiated breast cancer. We found that downregulation of IGF-IR results in tumor-size-dependent regression to an undetectable state. Partially regressed tumors almost always resumed growth in the absence of doxycycline and a proportion of tumors that regressed to an undetectable state ultimately recurred. This re-emergence of tumor growth in the absence of doxycycline was facilitated by IGF-IR-dependent and IGF-IR-independent mechanisms. Tumor escape from IGF-IR dependence was associated with an epithelial to mesenchymal transition and upregulation of transcriptional repressors of E-cadherin. These results suggest that tumors initiated by IGF-IR have the ability to become independent of this initiating oncogene, and IGF-IR independence is associated with characteristics consistent with an epithelial to mesenchymal transition.

Transgenic IGF-IR overexpression induces mammary tumors with basal-like characteristics, whereas IGF-IR-independent mammary tumors express a claudin-low gene signature

Molecular profiling has allowed a more precise classification of human cancers. With respect to breast cancer, this approach has been used to identify five subtypes; luminal A, luminal B, HER2-enriched, basal-like and claudin-low. In addition, this approach can be used to determine the type of tumor represented by particular cell lines or transgenic animal models. Therefore, this approach was utilized to classify the mammary tumors that develop in MTB-IGFIR transgenic mice. It was determined that the primary mammary tumors, which develop due to elevated expression of the type I insulin-like growth factor receptor (IGF-IR) in mammary epithelial cells, most closely resemble murine tumors with basal-like or mixed gene expression profiles and with human basal-like breast cancers. Downregulation of IGF-IR transgene in MTB-IGFIR tumor-bearing mice leads to the regression of most of the tumors, followed by tumor reappearance in some of the mice. These tumors that reappear following IGF-IR transgene downregulation do not express the IGF-IR transgene and cluster with murine mammary tumors that express a mesenchymal gene expression profile and with human claudin-low breast cancers. Therefore, IGF-IR overexpression in murine mammary epithelial cells induces mammary tumors with primarily basal-like characteristics, whereas tumors that develop following IGF-IR downregulation express a gene signature that most closely resembles human claudin-low breast tumors.

Mammary tumors that become independent of the type I insulin-like growth factor receptor express elevated levels of platelet-derived growth factor receptors

BackgroundTargeted therapies are becoming an essential part of breast cancer treatment and agents targeting the type I insulin-like growth factor receptor (IGF-IR) are currently being investigated in clinical trials. One of the limitations of targeted therapies is the development of resistant variants and these variants typically present with unique gene expression patterns and characteristics compared to the original tumor.ResultsMTB-IGFIR transgenic mice, with inducible overexpression of the IGF-IR were used to model mammary tumors that develop resistance to IGF-IR targeting agents. IGF-IR independent mammary tumors, previously shown to possess characteristics associated with EMT, were found to express elevated levels of PDGFRα and PDGFRβ. Furthermore, these receptors were shown to be inversely expressed with the IGF-IR in this model. Using cell lines derived from IGF-IR-independent mammary tumors (from MTB-IGFIR mice), it was demonstrated that PDGFRα and to a lesser extent PDGFRβ was important for cell migration and invasion as RNAi knockdown of PDGFRα alone or PDGFRα and PDGFRβ in combination, significantly decreased tumor cell migration in Boyden chamber assays and suppressed cell migration in scratch wound assays. Somewhat surprisingly, concomitant knockdown of PDGFRα and PDGFRβ resulted in a modest increase in cell proliferation and a decrease in apoptosis.ConclusionDuring IGF-IR independence, PDGFRs are upregulated and function to enhance tumor cell motility. These results demonstrate a novel interaction between the IGF-IR and PDGFRs and highlight an important, therapeutically relevant pathway, for tumor cell migration and invasion.

Characterization of a Novel Primary Mammary Tumor Cell Line Reveals that Cyclin D1 Is Regulated by the Type I Insulin-Like Growth Factor Receptor

The importance of type I insulin-like growth factor receptor (IGF-IR) overexpression in mammary tumorigenesis was recently shown in two separate transgenic models. One of these models, the MTB-IGFIR transgenics, was generated in our lab to overexpress IGF-IR in mammary epithelial cells in a doxycycline (Dox)-inducible manner. To complement this transgenic model, primary cells that retained Dox-inducible expression of IGF-IR were isolated from a transgenic mammary tumor. This cell line, RM11A, expressed high levels of IGF-IR, phosphorylated Akt, and phosphorylated extracellular signal–regulated kinase 1/2 in the presence of Dox. IGF-IR overexpression provided the primary tumor cells with a survival advantage in serum-free media and seemed to induce ligand-independent activation of the IGF-IR because RM11A cells cultured in the presence of Dox were largely nonresponsive to exogenous IGFs. IGF-IR overexpression also augmented the growth of RM11A cells in vivo because injection of these cells into mammary glands of wild-type mice produced palpable tumors in 15.8 ± 3.4 days when the mice were administered Dox, compared with 57.8 ± 6.3 days in the absence of Dox. DNA microarray analysis revealed a number of genes regulated by IGF-IR, one of which was cyclin D1. Suppression of IGF-IR expression in vitro or in vivo was associated with a decrease in cyclin D1 protein, suggesting that at least some of the proliferative actions of IGF-IR are mediated through cyclin D1. Therefore, this article characterizes the first primary murine mammary tumor cell line with inducible IGF-IR expression. These cells provide a powerful in vitro/in vivo model to examine the function of IGF-IR in mammary tumorigenesis. (Mol Cancer Res 2008;6(5):819–28)

ErbB2 enhances mammary tumorigenesis, oncogene-independent recurrence and metastasis in a model of IGF-IR-mediated mammary tumorigenesis

BackgroundThe type I insulin-like growth factor receptor (IGF-IR) and ErbB2 (Her-2) are receptor tyrosine kinases implicated in human breast cancer. Both proteins are currently the subject of targeted therapeutics that are used in the treatment of breast cancer or which are in clinical trials. The focus of this study was to utilize our inducible model of IGF-IR overexpression to explore the interaction of these two potent oncogenes.ResultsErbB2 was overexpressed in our RM11A cell line, a murine tumor cell line that overexpresses human IGF-IR in an inducible manner. ErbB2 conferred an accelerated tumor onset and increased tumor incidence after injection of RM11A cells into the mammary glands of syngeneic wild type mice. This was associated with increased proliferation immediately after tumor cell colonization of the mammary gland; however, this effect was lost after tumor establishment. ErbB2 overexpression also impaired the regression of established RM11A tumors following IGF-IR downregulation and enhanced their metastatic potential.ConclusionThis study has revealed that even in the presence of vast IGF-IR overexpression, a modest increase in ErbB2 can augment tumor establishment in vivo, mediate resistance to IGF-IR downregulation and facilitate metastasis. This supports the growing evidence suggesting a possible advantage of using IGF-IR and ErbB2-directed therapies concurrently in the treatment of breast cancer.

The RNF146 and tankyrase pathway maintains the junctional Crumbs complex through regulation of angiomotin.

ABSTRACT The Crumbs complex is an important determinant of epithelial apical-basal polarity that functions in regulation of tight junctions, resistance to epithelial-to-mesenchymal transitions and as a tumour suppressor. Although the functional role of the Crumbs complex is being elucidated, its regulation is poorly understood. Here, we show that suppression of RNF146, an E3 ubiquitin ligase that recognizes ADP-ribosylated substrates, and tankyrase, a poly(ADP-ribose) polymerase, disrupts the junctional Crumbs complex and disturbs the function of tight junctions. We show that RNF146 binds a number of polarity-associated proteins, in particular members of the angiomotin (AMOT) family. Accordingly, AMOT proteins are ADP-ribosylated by TNKS2, which drives ubiquitylation by RNF146 and subsequent degradation. Ablation of RNF146 or tankyrase, as well as overexpression of AMOT, led to the relocation of PALS1 (a Crumbs complex component) from the apical membrane to internal puncta, a phenotype that is rescued by AMOTL2 knockdown. We thus reveal a new function of RNF146 and tankyrase in stabilizing the Crumbs complex through downregulation of AMOT proteins at the apical membrane. Summary: We describe a new role for RNF146 and tankyrase in maintaining the Crumbs complex and apical-basal integrity in epithelial cells through regulation of ADP-ribosylation and ubiquitin-dependent degradation of the angiomotin AmotL2.

Murine mammary tumor cells with a claudin-low genotype

BackgroundMolecular classification of human breast cancers has identified at least 5 distinct tumor subtypes; luminal A, luminal B, Her2-enriched, basal-like and claudin-low. The claudin-low subtype was identified in 2007 and is characterized by low expression of luminal differentiation markers and claudins 3, 4 and 7 and high levels of mesenchymal markers. Claudin-low tumors have a reported prevalence of 7-14% and these tumors have a poor prognosis.ResultsIn this study we report the characterization of several cell lines established from mammary tumors that develop in MTB-IGFIR transgenic mice. Two lines, RM11A and RJ348 present with histological features and gene expression patterns that resemble claudin-low breast tumors. Specifically, RM11A and RJ348 cells express high levels of the mesenchymal genes Zeb1, Zeb2, Twist1 and Twist2 and very low levels of E-cadherin and claudins 3, 4 and 7. The RM11A and RJ348 cells are also highly tumorigenic when re-introduced into the mammary fat pad of mice.ConclusionsMammary tumor cells established from MTB-IGFIR transgenic mice can be used as in vitro and in vivo model systems to further our understanding of the poorly characterized, claudin-low, breast cancer subtype.

Abstract A10: Oncogenomic analysis of IGF-IR driven mammary tumors identifies a potential cell of origin and mechanisms of tumor recurrence

Genetically engineered mice are valuable tools for understanding the genetic events that contribute to breast cancer development and progression. We have previously generated a doxycycline (Dox) inducible model of breast cancer, MTB-IGFIR, in which overexpression of the human type-I IGF receptor (IGF-IR) leads to the rapid induction of ER-mammary tumors with mixed luminal and basal features. Following Dox withdrawal and subsequent transgene downregulation, a subset of tumors escape IGF-IR dependence and spontaneously recur with evidence of an epithelial-mesenchymal transition (EMT). To gain insight into the molecular pathways that drive primary and recurrent tumor growth in MTB-IGFIR mice, we carried out gene expression profiling and DNA copy number analysis. Gene-set enrichment analysis (GSEA) revealed expression of an Akt oncogenic signature in primary tumors, consistent with downstream signaling mediated by IGF-IR. Interestingly, primary tumors also expressed a luminal progenitor gene signature suggesting a potential cell of origin. Accordingly, flow cytometric analysis of Dox-pulsed mice showed that high expression of the IGF-IR transgene was confined to EpCAM+/high Sca-1− cells, a putative ER- alveolar progenitor population. Recurrent tumors were found to express a mammary stem cell (MaSC) gene signature, which is consistent with their more mesenchymal phenotype as well as increased cell cycle pathway activity. While aCGH analysis identified few genomic changes in primary tumors, recurrent tumors exhibited gains and losses in known oncogenes and tumor suppressors including MET receptor, YAP1 and p16Ink4a/p19ARF. Integration of mRNA and copy number data confirmed altered expression of these genes at the transcriptional level. Taken together, these results suggest IGF-IR drives tumor initiation in an ER- luminal progenitor cell population in part through activation of the Akt pathway and that multiple genetic alterations contribute to the acquisition of oncogene independence and tumor relapse. Citation Format: Robert A. Jones, Craig I. Campbell, Katrina L. Watson, Roger A. Moorehead. Oncogenomic analysis of IGF-IR driven mammary tumors identifies a potential cell of origin and mechanisms of tumor recurrence. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A10.

IGF-IR Mediated Mammary Tumorigenesis Is Enhanced during Pubertal Development

Although breast cancer typically develops in women over the age of 40, it remains unclear when breast cancer initiating events occur or whether the mammary gland is particularly susceptible to oncogenic transformation at a particular developmental stage. Using MTB-IGFIR transgenic mice that overexpress type I insulin-like growth factor receptor (IGF-IR) in a doxycycline-inducible manner, mammary tumorigenesis was initiated at different developmental stages. Tumor multiplicity was significantly increased while tumor latency was significantly decreased when the IGF-IR transgene was expressed during pubertal development compared to post-pubertal transgene expression. Moreover, metastatic spread of mammary tumors to the lungs was approximately twice as likely when IGF-IR was overexpressed in pubertal mice compared to post-pubertal mice. In addition, engraftment of pubertal MTB-IGFIR mammary tissue into cleared mammary fat pads of pubertal hosts produced tumors more frequently and faster than engraftment into adult hosts. These experiments show that the mammary microenvironment created during puberty renders mammary epithelial cells particularly susceptible to transformation.