Robert A. Jones

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.

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)

Unique roles of Akt1 and Akt2 in IGF-IR mediated lung tumorigenesis

AKT is a serine-threonine kinase that becomes hyperactivated in a number of cancers including lung cancer. Based on AKTs association with malignancy, molecules targeting AKT have entered clinical trials for solid tumors including lung cancer. However, the AKT inhibitors being evaluated in clinical trials indiscriminately inhibit all three AKT isoforms (AKT1–3) and it remains unclear whether AKT isoforms have overlapping or divergent functions. Using a transgenic mouse model where IGF-IR overexpression drives lung tumorigenesis, we found that loss of Akt1 inhibited while loss of Akt2 enhanced lung tumor development. Lung tumors that developed in the absence of Akt2 were less likely to appear as discrete nodules and more frequently displayed a dispersed growth pattern. RNA sequencing revealed a number of genes differentially expressed in lung tumors lacking Akt2 and five of these genes, Actc1, Bpifa1, Mmp2, Ntrk2, and Scgb3a2 have been implicated in human lung cancer. Using 2 human lung cancer cell lines, we observed that a selective AKT1 inhibitor, A-674563, was a more potent regulator of cell survival than the pan-AKT inhibitor, MK-2206. This study suggests that compounds selectively targeting AKT1 may prove more effective than compounds that inhibit all three AKT isoforms at least in the treatment of lung adenocarcinoma.

BMS-754807 is cytotoxic to non-small cell lung cancer cells and enhances the effects of platinum chemotherapeutics in the human lung cancer cell line A549

BackgroundDespite advances in targeted therapy for lung cancer, survival for patients remains poor and lung cancer remains the leading cause of cancer-related deaths worldwide. The type I insulin-like growth factor receptor (IGF-IR) has emerged as a potential target for lung cancer treatment, however, clinical trials to date have provided disappointing results. Further research is needed to identify if certain patients would benefit from IGF-IR targeted therapies and the ideal approach to incorporate IGF-IR targeted agents with current therapies.MethodsThe dual IGF-IR/insulin receptor inhibitor, BMS-754807, was evaluated alone and in combination with platinum-based chemotherapeutics in two human non-small cell lung cancer (NSCLC) cell lines. Cell survival was determined using WST-1 assays and drug interaction was evaluated using Calcusyn software. Proliferation and apoptosis were determined using immunofluorescence for phospho-histone H3 and cleaved caspase 3, respectively.ResultsTreatment with BMS-754807 alone reduced cell survival and wound closure while enhancing apoptosis in both human lung cancer cell lines. These effects appear to be mediated through IGF-IR/IR signaling and, at least in part, through the PI3K/AKT pathway as administration of BMS-754807 to A549 or NCI-H358 cells significantly suppressed IGF-IR/IR and AKT phosphorylation. In addition of BMS-754807 enhanced the cytotoxic effects of carboplatin or cisplatin in a synergistic manner when given simultaneously to A549 cells.ConclusionsBMS-754807 may be an effective therapeutic agent for the treatment of NSCLC, particularly in lung cancer cells expressing high levels of IGF-IR.

High levels of dietary soy decrease mammary tumor latency and increase incidence in MTB-IGFIR transgenic mice

BackgroundEpidemiologic data indicates that Asian diets, which are high in soy protein, reduce a women’s risk of developing breast cancer. However, it has been difficult to dissociate the benefits of soy from other variables including environmental and lifestyle factors. Since prospective studies in humans would take decades to complete, rodent models provide a valuable research alternative.MethodsIn this study, MTB-IGFIR transgenic mice, which develop mammary tumors resulting from overexpression of the type I insulin-like growth factor receptor (IGF-IR), were utilized. MTB-IGFIR mice were fed a soy-based or casein-based diet throughout all stages of development to reflect soy exposure in Asian cultures. Mammary tumors were initiated at 2 different developmental stages by commencing IGF-IR transgene expression either during puberty or in adult mice.ResultsMTB-IGFIR mice fed a soy-based diet displayed increased tumor incidence and accelerated tumor onset compared to MTB-IGFIR mice fed a casein diet. Two markers of estrogen receptor signaling, Pgr and Areg, were elevated in mammary tissue from mice fed the soy diet compared to mice fed the casein diet suggesting that high levels of soy may promote mammary tumor development through acting as an estrogen receptor agonist. Mammary tumors from mice fed a soy diet more frequently expressed metaplastic markers such as cytokeratins 5 and 14 as well as p63 and displayed reduced lung metastases compared to mammary tumors from mice fed a casein diet.ConclusionsDiets consisting of very high levels of soy protein promote mammary tumor development and decrease tumor latency possibly through activating estrogen receptor signaling. Additional studies are required to determine whether a more moderate amount of dietary soy can inhibit oncogene-induced mammary tumorigenesis.

Re-expression of miR-200c suppresses proliferation, colony formation and in vivo tumor growth of murine claudin-low mammary tumor cells

Claudin-low breast cancer is a relatively rare breast cancer subtype. These cancers are typically ER−/PR−/HER2− and express high levels of mesenchymal genes as well as genes associated with inflammation, angiogenesis and stem cell function. In addition to alterations in gene expression, it was recently demonstrated that claudin-low breast cancers express very low levels of the miR-200 family of miRNAs. Given that each miRNA can regulate tens, hundreds or even thousands of genes, miRNAs are being evaluated as therapeutic targets. In this study we show that mammary tumors from MTB-IGFIR transgenic mice and cell lines derived from these tumors represent a model of human claudin-low breast cancer and murine claudin-low mammary tumors and cell lines express only very low levels of all five members of the miR-200 family. Reduced miR-200 family expression appears to be regulated via methylation as cells and tumors expressing low levels of miR-200 family members had higher levels of CpG methylation in a putative promoter region than tumors and cells expressing high levels of miR-200 family members. Re-expression of miR-200c in murine claudin-low mammary tumor cells inhibited tumor cell proliferation and colony formation in vitro and tumor growth in vivo. With respect to tumor growth in vivo, re-expression of miR-200c was associated with a reduction in tumor vasculature and expression of Flt1 and Vegfc. Therefore, miR-200c is an important regulator of mesenchymal tumor cell growth.

Preneoplastic changes persist after IGF-IR downregulation and tumor regression.

Despite our incomplete understanding of the function of the type I insulin-like growth factor receptor (IGF-IR) in tumorigenesis, IGF-IR targeting agents have entered clinical trials for the treatment of human cancers. Previously, we have shown that downregulation of IGF-IR transgene in mammary tumors in MTB-IGFIR transengic mice results in tumor regression in a majority of the mice and most of these mice do not develop recurrent mammary tumors. In this study, we examined mammary tissue of mice that did not develop recurrent tumors. Areas of tumor regression were visible macroscopically and microscopically these lesions contained cell debris, individual cells, lipofuscin and doxycycline crystals. Three of the 12 mice also presented with considerable lobuloalveolar development. The re-expression of the IGF-IR transgene in mammary tissue with stably regressed tumors resulted in the rapid re-emergence of mammary tumors, some of which seemed to originate from the regressed mammary lesions. Thus, despite stable tumor regression after IGF-IR downregulation, mammary tissue contained preneoplastic lesions and tumors rapidly re-appear upon re-overexpression of IGF-IR transgene. Therefore, IGF-IR-targeting agents may be effective at regressing mammary tumors expressing IGF-IR, but these agents will not completely eradicate all tumor cells or restore the mammary stromal environment.

The function and therapeutic targeting of anaplastic lymphoma kinase (ALK) in non-small cell lung cancer (NSCLC)

Lung cancer is the leading cause of death by cancer in North America. A decade ago, genomic rearrangements in the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase were identified in a subset of non-small cell lung carcinoma (NSCLC) patients. Soon after, crizotinib, a small molecule ATP-competitive ALK inhibitor was proven to be more effective than chemotherapy in ALK-positive NSCLC patients. Crizotinib and two other ATP-competitive ALK inhibitors, ceritinib and alectinib, are approved for use as a first-line therapy in these patients, where ALK rearrangement is currently diagnosed by immunohistochemistry and in situ hybridization. The clinical success of these three ALK inhibitors has led to the development of next-generation ALK inhibitors with even greater potency and selectivity. However, patients inevitably develop resistance to ALK inhibitors leading to tumor relapse that commonly manifests in the form of brain metastasis. Several new approaches aim to overcome the various mechanisms of resistance that develop in ALK-positive NSCLC including the knowledge-based alternate and successive use of different ALK inhibitors, as well as combined therapies targeting ALK plus alternative signaling pathways. Key issues to resolve for the optimal implementation of established and emerging treatment modalities for ALK-rearranged NSCLC therapy include the high cost of the targeted inhibitors and the potential of exacerbated toxicities with combination therapies.

The miR-200b/200a/429 cluster prevents metastasis and induces dormancy in a murine claudin-low mammary tumor cell line

ABSTRACT The miR‐200 family of microRNAs consisting of miR‐141, miR‐200a, miR‐200b, miR‐200c and miR‐429 are emerging as important regulators of breast cancer progression. This family of microRNAs maintain mammary epithelial identity and downregulation of miR‐200 expression has been associated with epithelial‐to‐mesenchymal transition in mammary tumors. Therefore, re‐expression of one or more miR‐200 family members in mammary tumor cells with mesenchymal characteristics may restore an epithelial phenotype including growth and metastasis suppression. To test this hypothesis, the miR‐200b/200a/429 cluster was re‐expressed in a murine claudin‐low cell line, RJ423. Re‐expression of the miR‐200b/200a/429 cluster in RJ423 cells significantly suppressed the expression of Vim, Snai1, Twist1, Twist2 and Zeb1, reverted RJ423 cells to a more epithelial morphology and significantly inhibited proliferation in vitro. Moreover, the miR‐200b/200a/429 cluster prevented lung metastasis in an experimental metastasis model and although tumor initiation was not prevented, re‐expression of the miR‐200b/200a/429 cluster induced a dormancy‐like state where mammary tumors failed to grow beyond ˜150mm3 or grew extremely slowly following intra‐mammary injection. These dormant tumors contained elevated levels of collagen and were highly vascularized. Therefore, re‐expression of the miR‐200b/200a/429 cluster in the claudin‐low mammary tumor cell line, RJ423, is sufficient to alter cell morphology, impair metastasis and induce tumor dormancy. HIGHLIGHTSmiR‐200 family members regulate mesenchymal gene expression and cell shape.Expression of miR‐200b/200a/429 impairs mammary tumor metastasis.miR‐200b/200a/429 reduces tumor incidence and induces tumor dormancy.

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.