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. Author manuscript; available in PMC: 2016 Mar 8.
Published in final edited form as: Cancer Res. 2014 Aug 4;74(19):5668–5679. doi: 10.1158/0008-5472.CAN-14-0970

IGF-1R inhibition in mammary epithelia promotes canonical Wnt signaling and Wnt1-driven tumors

Lauren M Rota 1, Lidia Albanito 1, Marcus E Shin 1, Corey L Goyeneche 1, Sain Shushanov 1, Emily J Gallagher 2, Derek LeRoith 2, Deborah A Lazzarino 1,*, Teresa L Wood 1,*
PMCID: PMC4782979  NIHMSID: NIHMS619781  PMID: 25092896

Abstract

Triple-negative breast cancers (TNBC) are an aggressive disease subtype which unlike other subtypes lack an effective targeted therapy. Inhibitors of the insullin-like growth factor receptor (IGF-1R) have been considered for use in treating TNBC. Here we provide genetic evidence that IGF-1R inhibition promotes development of Wnt1-mediated murine mammary tumors that offer a model of TNBC. We found that in a double transgenic mouse model carrying activated Wnt-1 and mutant IGF-1R, a reduction in IGF-1R signaling reduced tumor latency and promoted more aggressive phenotypes. These tumors displayed a squamal cell phenotype with increased expression of keratins 5/6 and β-catenin. Notably, cell lineage analyses revealed an increase in basal (CD29hi/CD24+) and luminal (CD24+/CD61+/CD29lo) progenitor cell populations, along with increased Nanog expression and decreased Elf5 expression. In these doubly transgenic mice, lung metastases developed with characteristics of the primary tumors, unlike MMTV-Wnt1 mice. Mechanistic investigations showed that pharmacological inhibition of the IGF-1R in vitro was sufficient to increase the tumorsphere-forming efficiency of MMTV-Wnt1 tumor cells. Tumors from doubly transgenic mice also exhibited an increase in the expression ratio of the IGF-II-sensitive, A isoform of the insulin receptor vs the IR-B isoform, which in vitro resulted in enhanced expression of β-catenin. Overall, our results revealed that in Wnt-driven tumors an attenuation of IGF-1R signaling accelerates tumorigenesis and promotes more aggressive phenotypes, with potential implications for understanding TNBC pathobiology and treatment.

Keywords: IGF-1R, β -catenin, Wnt, IGF-II, IR-A

Introduction

Triple negative breast cancers (TNBCs), classified as estrogen receptor- and progesterone receptor-negative and lacking HER2 amplification remain the most challenging breast tumor subtype to treat due to their aggressive nature and the lack of effective treatment options. A number of recent studies have focused on identifying receptors and signaling pathways active in TNBCs; these studies and other molecular profiling analyses have revealed considerable heterogeneity within this breast cancer subtype however, approximately 50% of TNBCs classify as basal-like (1). Interestingly, several pathways with known roles in embryonic development and stem/progenitor cell self-renewal have been identified with critical roles in TNBCs including the Wnt/β-catenin, Notch and Hedgehog pathways (for review see, (2)). In this study we used the MMTV-Wnt1 tumor mouse model, which forms tumors that predominantly express basal cell markers, are enriched for mammary stem and basal cell progenitors and have transcriptomic characteristics similar to those arising in BRCA1 germline mutation carriers (3-5).

The canonical Wnt pathway was first associated with mammary carcinogenesis when the Int-1 integration site of the mouse mammary tumor virus (MMTV) was identified as a mammalian homolog of Drosophila Wingless polarity morphogen and was renamed Wnt1 (6). In subsequent experiments, Varmus and colleagues further demonstrated that Wnt1 overexpression in mammary epithelium is sufficient to form tumors in mice (7). An important intracellular response to secreted Wnt1 is the stabilization of β-catenin, which can enter the nucleus and transactivate Wnt target genes. The stabilization of β-catenin is a hallmark of canonical Wnt signaling, which is enhanced in human basal-like breast cancers (8). A recent study further demonstrated an association of Wnt signaling with lung and brain metastases in TNBC patients (9).

Recent data suggest that the insulin-like growth factor (IGF) signaling axis also has a role in TNBC. IGF gene signatures are increased in TNBCs and TNBC cell lines, and IGF signaling promotes proliferation and survival of TNBC cells (10, 11). The IGF signaling system consists of two ligands, IGF-I and IGF-II, which can activate several receptor subtypes. Both ligands have high affinity for the IGF type I receptor (IGF-1R), which has been implicated in several types of cancer including prostate, colon and breast (12, 13). Results from early studies demonstrated that the IGF-1R is necessary for transformation of fibroblasts by a variety of oncogenes (for review, see (14). Subsequent studies demonstrated that either overexpression or constitutive activation of the IGF-1R in mammary epithelium results in hyperplasia and development of tumors (15, 16). In addition to the IGF-1R, IGF-II also can signal through the A isoform of the insulin receptor (IR-A), a splice variant of the IR that lacks exon 11. IGF-II signaling through the IR-A is important in embryonic development (17), and this signaling loop is also prevalent in a variety of cancers (18-20). The ratio of IR-A:IR-B is higher in breast cancer cell lines and in primary breast tumors than in normal tissue (19, 21). Compared to the IR-B isoform, which is the more common metabolic form of the IR found on insulin-sensitive cells, the IR-A functions in cell growth, proliferation and survival (For review, see (22)). Interestingly, phosphorylation and total levels of IR (but not IGF-1R) have been correlated with poor survival in patients with invasive breast cancer of all subtypes (23). These studies highlight the complexity of IGF signaling and the need for a better understanding of how it functions in the context of oncogene pathways.

Here, we tested the function of the IGF-1R in MMTV-Wnt-1 mediated mammary tumorigenesis by co-expressing a kinase-dead IGF-1R transgene under the control of the MMTV promoter. We demonstrate the attenuation of IGF-1R in combination with Wnt1 overexpression decreases mammary tumor latency and incidence, increases the basal cell and aggressive phenotype of the tumors and leads to lung metastases. Similarly, acute pharmacological inhibition of the IGF-1R is sufficient to increase tumorsphere-formation in vitro. We further demonstrate that the reduction in IGF-1R signaling in the MMTV-Wnt1 tumor model enhances an IGF-II/IR-A signaling loop that enhances canonical Wnt signaling.

Materials & Methods

Transgenic Mouse Lines

All animal protocols were approved by Rutgers University (formerly University of Medicine and Dentistry of New Jersey, UMDNJ) Institutional Animal Care and Use Committees (IACUC). All experiments were managed in accord with the National Institutes of Health guidelines for the care and use of laboratory animals. The MMTV-dominant-negative IGF-1R (dnIGF-1R) transgenic mouse line was established by pronuclear injections into FVB embryos. The MMTV-LTR promoter and the kinase-dead human IGF-1R used for the transgenic construct were described previously (24-26). Three independent transgenic lines were obtained, which were used to verify transgene expression and initial phenotype. For developmental analysis, hemizygous or homozygous males and females were mated to obtain transgenic or wild-type females for experiments. For flow cytometry, FBV age-matched wild-type females were used as controls. The MMTV-Wnt1 line on an FVB background (FVB.Cg-Tg(Wnt1)1Hev/J) was obtained from Jackson Laboratories. The MMTV-Wnt1//MMTV-dnIGF-1R (bigenic) line was obtained by crossing a MMTV-Wnt1 hemizygous male with a MMTV-dnIGF-1R homozygous female. Female littermates (bigenic vs MMTV-Wnt1) were used for experiments. Animal care was provided by the veterinary staff of the division of animal resources in the New Jersey Medical School Cancer Center of Rutgers Biomedical Health Sciences. MMTV-Wnt1 and bigenic female mice were palpated every five days for tumors beginning at three weeks of age; tumors were harvested when they reached one centimeter.

Mammary epithelial cell purification, flow cytometry and cell culture

Primary MECs were purified from non-malignant, pre-malignant and malignant mammary glands as described previously, except tumor cells were enzymatically digested for 2.5 hrs (27). For mammary cell sorting, single MEC cell suspensions from non-malignant and premalignant tissue were obtained from the thoracic and inguinal mammary glands (28, 29).

Cytometric analysis of cells immunolabeled with fluorochrome-conjugated surface labeling and/or intracellular labeling was performed as described (28). Hematopoietic lineage markers (PE-conjugated anti mouse CD45, CD140a, Gr-1, TER119, CD31, and CD11c) (Biolegend) were used to exclude stromal and hematopoietic cell contamination (lineage minus, Lin-). Acquisition and analysis of cell-associated fluorescence were performed using a BD FACSCalibur® cytometer or LSR and TreeStar Inc. FlowJo® software, respectively.

For FACS, antibodies and mammary epithelial single cells were resuspended at 106 cells/ml in PBS with 2% BSA and 2% goat serum (FACs buffer). Cells were immunolabeled with fluorochrome-conjugated anti- CD29 Alexa Fluor 647 (1:280 Biolegend), CD24 Alexa Fluor 488 (1:250 Biolegend), and CD61 Biotin (1:200 BD Pharmigen). Single cells were prepared for FACs as previously described (30). Cells were sorted at 70psi using a 70 micron nozzle on the Becton Dickinson FACS Vantage™.

The sphere limiting dilution assay (SLDA) was performed as described previously (31). SLDA experiments for tumorspheres utilized the functional blocking antibody, IMC-A12 (15 μg/ml), a monoclonal antibody against IGF-1R (provided by ImClone Systems, a wholly owned subsidiary of Eli Lilly and Co).

For signaling experiments, mammary organoids were isolated and treated with 50 nM of IGF-1 for 15 min at 37°C and then harvested for Western Blot or RT-PCR analysis as described previously (32, 33). The IGF-1R null (R-) IR-A overexpressing mouse fibroblasts were established by transfecting human IR-A cDNA into R- cells, derived from mice with a targeted disruption of the igf-1r gene (34-36). The R-/IR-A cell line was obtained from A. Morrione (Thomas Jefferson University, Philadelphia, PA) two months prior to use and were re-tested by PCR one month prior to use to confirm that they lacked the mouse IGF-1R and expressed human IR-A.. Cells were starved in serum and phenol red free media for 24 hours prior to exogenous treatment with IGF-II. IGF-II was added to serum free media in doses from 10-200 nM and time periods from 20 min to 24 hours.

RNA Isolation and Quantitative Real-Time PCR

Freshly isolated non-malignant, premalignant and tumor samples were used for RNA extraction with TRIzol (GIBCO) according to manufacturer's instructions. For real-time PCR, total RNA (500 ng) from Universal mouse reference RNA (Stratagene, La Jolla, CA) and primary cells (normal and tumor mammary tissue) were reverse- transcribed as previously described (32). Samples were run in triplicate and results were analyzed on ABI 7400 software. β-actin was used as the endogenous control. Mouse primers for Twist1, Nanog, Hey1, DLL4, Elf-5 and human IGF-1R were from Quantitect. The primers used to analyze IR-A and IR-B were described previously (32).

Histology and immunohistochemistry

Tissue samples were drop-fixed in 3% PFA for six hours and embedded in paraffin and sectioned at 10 μm. Primary tumor and lung sections were used for hematoxylin and eosin staining or further processed for antigen retrieval and immunofluorescence (IF) (33). Primary antibodies included cytokeratin 5, (1:200, Covance); β-catenin (1:200, Santa Cruz); cytokeratin 6 (1:1000, Covance); secondary antibodies and DAPI were as previously described (33). Lung metastases were counted in 3 H&E stained sections per lung, 80 μm apart by an investigator blinded to genotype (n=4).

Protein Isolation and Western immunoblotting

Total protein was isolated and used for Western immunoblotting as described previously (32) using antibodies to β-catenin (1:1000; Santa Cruz Biotechnology), P-Akt (Ser473) (3787), Akt (9272), P-Erk 1/2 (Thr 202 and Tyr 204) and total Erk (1:1000, all from Cell Signaling Technology). β-actin (1:5000; Sigma) was used as an endogenous control.

Statistical analyses

Statistical differences for RT-PCR, western immunoblotting and flow cytometry analyses were determined using Student's t test for two group comparisons. P values < 0.05 were used to represent significance. All results were reproduced across multiple experiments. Statistical significance for the Kaplan Meier data was determined using a Mantel Cox test and Graph pad software.

Results and Discussion

Attenuated IGF-1R signaling in the MMTV-Wnt1 mammary tumor model decreases tumor latency and increases tumor multiplicity

To investigate the role of IGF-1R signaling in Wnt1-mediated mammary tumorigenesis, we established a bitransgenic (bigenic) mouse line carrying a kinase-dead, dominant-negative IGF-1R transgene driven by the MMTV promoter (MMTV-dnIGF-1R) and a MMTV-Wnt1 transgene. Nulliparous bigenic female mice developed mammary tumors with decreased latency compared to the MMTV-Wnt1 female mice (Figure 1A). MMTV-Wnt-1 expressing mice had a mean latency consistent with previous results (37, 38). No mammary tumors were detected in mice carrying only the MMTV-dnIGF-1R transgene up to 1 year. The bigenic mice also showed increased tumor multiplicity; 12 of 16 bigenic females developed more than 1 tumor whereas only 1 of 13 MMTV-Wnt1 females developed more than one tumor (Figure 1B). Growth rates of tumors measured from time of palpation to harvest were similar between the MMTV-Wnt1 and bigenic strains (Figure 1C).

Figure 1. Decreased tumor latency and increased squamous cell phenotype in bigenic mice compared to Wnt1 alone mice.

Figure 1

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A, Kaplan–Meier curve showing tumor latency in the MMTVWnt1//MMTV-dnIGF-1R (bigenic) mouse line compared to that of the MMTV-Wnt1 (Wnt1) mouse line (n=21 per genotype; Log-rank Mantel-Cox test p=0.02). B, Graph showing tumor incidence in bigenic mice vs. Wnt1 mice. C, Graph showing tumor growth rates for bigenic and Wnt1 mammary tumors. D-E, Representative H&E stained sections from Wnt1 (D) and bigenic (E) tumors. F-G, Representative photomicrographs showing immunofluorescence staining for cytokeratin 5 (K5; red) and β-catenin (b-cat; green) in Wnt1 (F) and bigenic (G) tumors. H-I, Representative photomicrographs showing immunofluorescence staining for cytokeratin 6 (K6; red) in the Wnt1 (H) and bigenic (I) tumors. Sections in F-I were stained for DAPI to detect nuclei (blue). Size bar in D = 100 μm (for panels D-I).

Histopathology of the mammary tumors revealed an increase in squamous and pillar cells in the bigenic tumors compared to MMTV-Wnt1 mammary tumors (Figure 1D,E). A similar increase of squamal differentiation was shown previously in a mouse line expressing a stabilized β-catenin transgene (ΔE3 β-catenin) (39, 40). The bigenic tumors showed increased staining for both cytokeratin 5 (K5) and β-catenin (Figure 1F,G). Expression of cytokeratin 6 (K6), a marker of mammary biopotential progenitor cells (41) and expressed in MMTV-Wnt1 tumors (42), was also more prevalent in the bigenic tumors compared to Wnt1 tumors (Figure 1H,I). Increased expression of K6 was also observed in the ΔE3 β-catenin tumors where it was used as a marker of squamal cell differentiation (40). These data suggest that decreased IGF-1R signaling accelerates tumor initiation and alters tumor characteristics towards a more basal and squamal cell phenotype in the context of elevated Wnt signaling.

Loss of IGF-1R during normal mammary development increases luminal progenitors and decreases stem/myoepithelial lineages

IGF-1R signaling functions in proliferation and survival in a variety of cell types and has been recognized as a promoter of tumorigenesis when overexpressed in multiple tissues, including mammary epithelium. However, our findings suggest the attenuation of the IGF-1R in vivo has the opposite effect in the context of high Wnt signaling. These unexpected results suggested the possibility that the expression of the dnIGF-1R transgene may have altered normal mammary epithelial development making it more susceptible to Wnt1 tumor initiation. To test this hypothesis, we examined the effect of the MMTV-dnIGF-1R transgene on normal epithelial cell populations in early development in the absence of Wnt1 expression. Morphological analyses of the MMTV-dnIGF-1R glands by whole mount staining revealed a reduction in tertiary branching in post-pubertal glands (Figure 2A-D). Expression of the dnIGF-1R transgene was verified using primers to the human IGF-1R (Figure 2E). To confirm that IGF signaling was reduced in mammary epithelial cells of the MMTV-dnIGF-1R transgenic line, we treated isolated primary mammary organoids with IGF-I to stimulate IGF-1R signaling. IGF-I stimulation in the wild-type MECs led to increased levels of P-Akt and P-Erk1/2 as expected (p<0.001); however, levels of P-Akt and P-Erk1/2 were reduced in MECs from MMTV-dnIGF-1R glands (p≤0.02; Figure 2F,G).

Figure 2. dnIGF-1R mammary glands have decreased glandular development and cell lineage alterations.

Figure 2

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A-D, representative whole mount staining of wild-type (WT; A,C) and MMTV-dnIGF-1R (B,D) mammary glands from two different animals. E, Expression of the human IGF-1R transgene was detected by RT-PCR in MMTV-dnIGF-1R MECs (DN), bigenic hyperplasia (BI-hyp), and bigenic tumors (BI-tum). Mouse universal cDNA (Ms+) was used as a negative control. F-G, Graphs showing levels of P-Akt/total Akt (F) or P-Erk/total Erk (G) following acute stimulation of purified mammary epithelial spheroids with IGF-I. Western immunoblotting was performed on protein isolated from epithelial preps from each genotype (n=3). *p<0.001 vs control, **p≤0.01 vs WT. H-I, Flow cytometry using mammary lineage markers CD29 APC, CD24 FITC, and excluding lineage markers (CD45, CD31, CD11b, Gr-1, Ter119) of purified MECs from WT and MMTV-dnIGF-1R glands at 11 weeks. CD24+ CD29hi in MMTV-dnIGF-1R versus WT: p<0.001; CD24+ CD29lo in dnIGF-1R versus Wt: p<0.001. J-K, Flow cytometry using CD24+ CD29lo CD61+ lin- cell markers for the identification of the luminal progenitor population. CD24+ CD29lo CD61+ lin- population in the MMTV-dnIGF-1R MECs versus WT: p<0.001 (n=4 independent flow cytometry analyses for H-K). L-M, RT-PCR analysis of Hey1 (L) and DLL4 (M) expression in 10 week MMTV-dnIGF-1R MECs versus 10 week WT MECs (L, *p=0.05; M, *p≤0.02).

The reduction in tertiary branching of glands derived from the MMTV-dnIGF-1R mice, suggested the possibility that the reduction in IGF signaling had altered proliferation and or differentiation in specific epithelial cell lineages (43). We previously showed that the expression of the MMTV-dnIGF-1R reduced mammosphere forming potential in a sphere limiting dilution assay (SLDA) in vitro suggesting a reduction in the basal cell lineage (31). To directly analyze lineage alterations, we isolated MECs from MMTV-dnIGF-1R and wild-type glands and analyzed the expression of lineage specific markers by flow cytometry, using antibodies to the cell surface markers CD24 (heat shock antigen) and CD29 (β1 integrin) which distinguishes luminal (CD24+/CD29lo) from stem/ basal cell populations (CD24+/CD29hi) (28, 29). Flow cytometry analysis of MMTV-dnIGF-1R epithelial cells revealed an increase in the luminal (CD29lo /CD24+ Lin-) population (p<0.001; Figure 2 H-I) and a two-fold decrease in the basal population (CD24+CD29hi Lin-) (p<0.001; Figure 2H-I). The inclusion of CD61 (β3 integrin), in combination with CD24 and CD29, revealed a two-fold expansion in the luminal progenitor population in the MMTV-dnIGF-1R epithelium (p<0.001; Figure 2J-K) (44). The shifts in cell lineage populations were observed in MMTV-dnIGF-1R glands from both normal cycling animals and in animals synchronized with a E2/P4 injections (data not shown).

The results of the flow cytometry analyses support the hypothesis that a shift in cell lineage may be responsible for the hypo-branching phenotype as well as the enhanced Wnt tumor susceptibility in the MMTV-dnIGF-1R glands. Macias et al. (2011) showed that a reduction in basal cells reduces the numbers of lateral branches (43). Importantly, several studies demonstrate that mammary progenitor cells are the targets of oncogenesis by the Wnt pathway (42, 45, 46) including a luminal progenitor population (46). Thus, expansion of the luminal progenitor population due to a reduction in IGF signaling may lead to increased susceptibility to Wnt-mediated transformation. In support of this is the finding that luminal progenitors are the likely targets for transformation in BRCA1 tumors (47).

Based on the developmental and lineage alterations in the MMTV-dnIGF-1R glands, we hypothesized that defective IGF signaling may have enhanced another signaling pathway responsible for luminal progenitor expansion. A likely candidate for this is the Notch signaling pathway; constitutive activation of Notch signaling promotes expansion of undifferentiated CD61+ CD29lo luminal progenitors (48). Moreover, Notch signaling is necessary for Wnt1 mediated tumorigenesis in human mammary epithelial cells (49). Expression of the Notch target gene Hey1 and the Notch ligand Dll4 were increased in primary MECs from MMTV-dnIGF-1R glands (p≤0.05; Figure 2L-M). Taken together, our findings support the hypothesis that the increased numbers of luminal progenitors and susceptibility to Wnt-mediated tumors may be due to dysregulation of Notch signaling in the dnIGF-1R epithelium.

Increased Notch signaling in dnIGF-1R/Wnt1 hyperplasia

Because we observed a disruption in lineage distribution in the MMTV-dnIGF-1R mammary epithelium, we analyzed premalignant epithelium to determine if the myoepithelial to luminal progenitor lineage shift persisted in hyperplasia of the bigenic glands. We isolated tumor-free, pre-neoplastic glands contralateral to glands containing tumors. Since most tumors developed at an earlier time in the bigenic mice versus the MMTV-Wnt1 mice, the hyperplastic epithelium was stage-equivalent but not age-matched. The luminal and basal populations appeared similar in the stage-matched hyperplasia between the two genotypes; specifically the CD61+CD29lo luminal progenitor populations were similar between MMTV-Wnt1 and bigenic glands (Figure 3A-D). Since mammary tumors developed as early as 7-8 weeks in the bigenic mice, we analyzed age-matched glands at 5 weeks from both genotypes, a time when both lines were tumor-free. Bigenic and MMTV-Wnt1 glands at 5 weeks were both hyperplastic and showed no distinct morphological differences (Figure 3E-F). Although the MMTV-Wnt1 tumors have a predominant basal phenotype, they also contain a population of luminal-type cells with some expression of ER and PR (5, 50). Thus, we measured RNA expression of progesterone receptor (PR) and RANKL in MMTV-Wnt1 compared to bigenic hyperplasia in luminal epithelial fraction after FACs. PR and RANKL expression were unchanged between the two tumor types (data not shown). Despite the absence of cell lineage changes, morphological alterations, or hormone receptor status and signaling, the bigenic hyperplasia had a significant increase in expression of the Notch target gene, Hey1, (p≤0.05; Figure 3G). The increase in Notch signaling in bigenic hyperplasia despite no difference in the size of the progenitor populations suggests an amplification of Notch signaling in individual progenitor cells. The lack of detectable amplification of the luminal progenitor population in the bigenic vs MMTV-Wnt1 hyperplasia may be due to the ability of the Wnt pathway alone to amplify this population. These data suggest that the increase in Notch signaling that persists in the bigenic hyperplasia may expedite tumor formation.

Figure 3. Stage matched hyperplasia from MMTV-Wnt1 and bigenic glands have similar lineage distributions.

Figure 3

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A-B, CD24 FITC and CD29 APC Lineage negative (CD45, CD31, CD11b, Gr-1 Ter119) FAC sorted mammary cells from MMTV-Wnt1 (A) and bigenic (B) hyperplastic mammary glands. C-D, CD24 FITC, CD29 APC and CD61 PerCP Cy5 Lineage negative FAC sorted cells in MMTV-Wnt1 (C), and bigenic (D) hyperplasia. A-D are representative of four independent sorts. E-F, whole mounts of MMTV-Wnt1 (E) and bigenic (F) abdominal mammary glands taken at 5-weeks of age. G, RT-PCR analysis of Hey1 expression in MMTV-Wnt1 and bigenic hyperplasia (*p≤0.05).

Disruption of the IGF-1R in the MMTV-Wnt1 tumor model enhances the basal tumor phenotype and increases the luminal progenitor population

Although there were no obvious morphological or lineage differences in the hyperplasia, there were clear morphological differences in the bigenic versus MMTV-Wnt1 tumors (Figure 1). When we analyzed the tumor cell populations by flow cytometry, we found that the bigenic tumors had an increase in the basal cell population (p<0.001) and a decrease in the luminal cell population (p<0.001) (Figure 4A,B). Furthermore, we found an increase in the CD61+CD24+CD29lo luminal progenitor population in the bigenic tumors compared to the Wnt1 tumors (p=0.01; Figure 4C,D). Interestingly, this population was previously described as the putative tumor initiating population in an MMTV-Wnt1 mammary tumor model (46, 51), initially shown by Li et. al. (42). These results suggest a shift towards a more basal tumor phenotype in MMTV-Wnt1 tumors in the presence of the dnIGF-1R transgene. Similar to analysis of the hyperplasia, PR and ER expression were unchanged in the MMTV-Wnt1 compared to the bigenic tumors (data not shown).

Figure 4. Bigenic tumors have an increased basal cell phenotype.

Figure 4

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A-B, CD24-Alexa Fluor® 488- and CD29-postive and Alexa Fluor® 647-positive, Lineage-negative (CD45, CD31, CD11b, Gr-1 Ter119), FAC-sorted cells from MMTV-Wnt1 (A) and bigenic (B) tumors. Dot plots show the differences observed for CD24+ CD29hi basal tumor cells (p<0.001) and luminal tumor cells (p≤0.001). C-D, CD24-Alexa Fluor® 488, CD29-Alexa Fluor® 647and CD61-PerCP-Cy5 in Lin-FAC-sorted cells from MMTV-Wnt1 (C) and bigenic (D) tumors. Dot plots show the differences observed for CD24+ CD29lo CD61+ luminal progenitors (p≤0.01). (n=5 independent sorts for A-D). E-F, Changes in Elf-5 (E) and Nanog (F) expression between MMTV-Wnt1 vs bigenic tumors were determined by RT-PCR. Elf5, *p≤0.05 (n=3); Nanog, *p≤0.05 (n=5).

We further analyzed the MMTV-Wnt1 and bigenic tumors for changes in expression of markers of differentiation and epithelial to mesenchyme transition (EMT). Elf-5, which has known roles in alveologenesis as well as in mammary stem and progenitor cell fate (52) was decreased in bigenic tumors compared to MMTV-Wnt1 tumors (p≤0.05; Figure 4E). Targeted deletion of Elf-5 in mice leads to an accumulation of undifferentiated luminal and basal progenitors (53), and loss of Elf-5 leads to an increase in the CD61+ luminal progenitors and hyperactive Notch signaling (52, 53). Finally, conditional knockouts of Elf-5 in the MMTV-Neu tumor model showed that Elf-5 represses markers of EMT (53).

Since the bigenic tumors showed an increase in the basal cell population which would confound interpretation of alterations in expression of EMT genes, we isolated RNA from FAC-sorted basal and luminal populations, from bigenic vs MMTV-Wnt1 tumors. Expression of Twist 1 and Nanog, genes that are upregulated in EMT (54), was increased in the bigenic tumor basal population; the increase in Nanog expression was statistically significant (p≤0.05, Figure 4F). Elevation of Twist 1 approached statistical significance (p=0.06, data not shown). Expression of these genes was unaltered in the luminal cell population (data not shown). Collectively, these data suggest that loss of IGF-1R signaling in the presence of an over-expression of the Wnt1 oncogene enhances the basal phenotype of the resulting tumor and increases the putative metastatic potential by attenuating Elf-5 expression and increasing EMT mediators.

Increased lung metastases in bigenic mice

The more aggressive tumor phenotype in the bigenic mice suggested the possibility that they might more readily contribute to lung metastases. Typically MMTV-Wnt1 tumors, although basal-like, are not highly metastatic and produce few lung metastasis (7). The bigenic mice had detectable lung metastases (Figure 5A,B; 3.4 mets/lung) whereas the MMTV-Wnt1 mice had no detectable metastases at the time of primary tumor removal (n=4/genotype). Consistent with expression of K5 and K6 in the primary bigenic tumors, bigenic lung metastases also showed K5 and K6 expression (Figure 5C-F). K5 staining appeared also in basal layers around some aveoli in the lungs from both genotypes as well as wild-type lungs (not shown). However, no K5 was seen in other areas of the MMTV-Wnt1 lungs, and we observed no detectable K6 staining in either the MMTV-Wnt1 or wild-type lungs. The K6 staining was absent from the smaller metastases (Figure 5E) but was prevalent in large lung metastases of the bigenic animals (Figure 5F) suggesting that the primary tumor cells express K6 after transdifferentiation to a more squamal phenotype.

Figure 5. Increased lung metastasis in bigenic mice.

Figure 5

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A-B, Representative H&E stained sections from lungs taken from bigenic mice with tumors showing a small (A) and large (B) metastasis. C-E, Representative photomicrographs showing immunofluorescence staining for K5 (C,D) and K6 (E,F) in metastases of the bigenic mice. Sections in C-F were stained for DAPI to detect nuclei (blue). Size bar in A = 100 μm (for all panels).

Pharmacological inhibition of IGF-1R signaling in Wnt1 primary tumor cells in vitro increases tumorsphere formation

We hypothesized that the alteration in phenotype of the bigenic tumors could be due to ongoing expression of the dnIGF-1R transgene. Analysis of the bigenic hyperplasia and tumors showed transgene expression equivalent to the expression in the MMTV-dnIGF-1R glands in the absence of the MMTV-Wnt1 transgene (Figure 2E). To test if acutely inhibiting IGF-1R in primary MMTV-Wnt1 tumor cells in vitro leads to a more aggressive growth phenotype, we treated MMTV-Wnt1 tumor cells with an IGF-1R blocking antibody in a SLDA (31, 55). When IGF-1R signaling was inhibited with a blocking antibody (A12), we observed an increase in frequency of tumorsphere formation by the MMTV-Wnt1 tumor cells from 1 in 101 to 1 in 24 (Figure 6A). Moreover, we sorted primary MMTV-Wnt1 tumor cells using basal and luminal lineage cell markers and measured sphere forming frequency in both luminal and basal MMTV-Wnt1 tumor cells in tertiary spheres plus or minus the IGF-1R blocking antibody. As expected, the basal cell population showed a greater tumor initiating frequency in the absence of IGF-1R inhibition (1 in 83 for MMTV-Wnt1 basal cells and 1:345 for MMTV-Wnt1 luminal cells). In the presence of the IGF-1R blocking antibody, both luminal and basal sorted primary MMTV-Wnt1 tumor cells showed enhanced tumorsphere forming frequency (1 in 52 and 1 in 40, respectively; Figure 6B,C). Interestingly, IGF-1R inhibition altered the tumorsphere forming potential of the luminal cells to a level similar to that of the basal population. Studies using an MMTV-Neu mouse model have demonstrated that the frequency of tumorsphere formation strongly correlates to the number of tumor initiating cells (TIC) in a given population (56, 57). TIC potential does not necessarily correlate to the cell of origin, but rather is an indication of the ability of a cell to self-renew and initiate tumorigenesis (55). Thus, the increase in tumorsphere growth by the MMTV-Wnt1 tumor cells with IGF-1R inhibition suggests that attenuating IGF-1R signaling enhances the TIC population of MMTV-Wnt1 tumors.

Figure 6. Pharmacological inhibition of IGF-1R increases tumorsphere forming frequency of MMTV-Wnt1 tumor cells.

Figure 6

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Tumorsphere limiting dilution analysis in tertiary spheres of freshly isolated MMTV-Wnt1 tumor cells treated with a monoclonal antibody inhibitor of the IGF-1R (A12) vs control IgG antibody. Linear regression analysis (linear Wnt; linear Wnt + A12) was performed to determine sphere forming frequency. A, tertiary tumorspheres from a MMTV-Wnt1 tumor treated plus or minus A12. B-C, MMTV-Wnt1 tumor cells were FAC sorted for CD24+CD29hi Lin- cell population (B) or CD24+CD29lo Lin- cell population (C) and plated in a limiting dilution tumorsphere assay plus or minus A12 IGF-1R antibody.

Attenuation of the IGF-1R in MMTV-Wnt1 mammary epithelium increases IR A:IR-B expression and canonical Wnt signaling

We observed a decrease in tumor latency, enhanced basal phenotype and squamous cell transdifferentiation in the bigenic mice. In addition, tumorsphere-forming frequency of MMTV-Wnt1 tumor cells increased when IGF-1R signaling was attenuated in vitro. Taken together, these findings suggest that the canonical Wnt pathway may be enhanced in the absence of IGF signaling. Consistent with this hypothesis, protein levels of β-catenin, normally stabilized by activation of canonical Wnt signaling, were increased in the bigenic tumors vs Wnt-1 tumor cells (Figure 7A).

Figure 7. Increased β-catenin levels and enhanced IGF-II and IR-A profiles in bigenic tumors.

Figure 7

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A, Western immunoblotting showing levels of β-catenin protein in MMTV-Wnt1 and bigenic tumors (n=3). B-C, RT-PCR for IR-A and IR-B was performed using a previously published assay (32). Graphs show ratio of IR-A:IR-B isoforms in (B) MMTV-Wnt1 and bigenic tumors (n=3; p<0.01) or (C) in MMTV-Wnt1 and bigenic FAC-sorted luminal hyperplasia cells (CD24+CD29lo) (n=3; p<0.002). D, Graph showing quantification from Western immunoblotting for levels of phospho-IRS-1/total IRS-1 in MMTV-Wnt1 and bigenic tumors (n=3; p=0.05). E, RT-PCR quantification of IGF-II expression in MMTV-Wnt1 and bigenic tumors (n=4; p=0.05). D, β-catenin levels in IGF-1R null (R-) fibroblast cells over-expressing IR-A and treated with increasing concentrations of IGF-II. Graph shows quantification of β-catenin levels following IGF-II treatment of R-/IR-A fibroblasts. 10 nM (p=0.18) 50 nM (*p=0.05) and 100 nM (**p=0.008).

Down-regulation of IGF-1R signaling in a variety of breast cancer cell lines increases insulin sensitivity by increasing cell surface expression of holo-IR because of a reduction in hybrid IR/IGF-1R cell surface expression (58). Moreover, MMTV-Wnt1 tumors are responsive to diet-induced obesity as well as caloric restriction suggesting insulin has a role in MMTV-Wnt1 tumor growth (59). We hypothesized that the reduction of IGF-1R in the context of Wnt1 signaling may have led to an alteration in IR signaling. When we determined the expression of IR isoforms IR-A and IR-B, we saw a significant increase in the IR-A:IR-B ratio in the bigenic tumors (p<0.01: Figure 7B). The increase in the ratio of IR-A to IR-B was due predominantly to a decrease in IR-B expression, consistent with findings in other tumors (21, 60). To determine if the increase in IR-A:IR-B occurred prior to tumor onset, we determined the expression of the IR isoforms in the FAC-sorted luminal hyperplasia population (CD24+CD29lo), since the luminal population accounted for the majority of the hyperplastic epithelium. We found that the IR-A:IRB ratio was significantly enhanced in the bigenic luminal hyperplasia despite no change in cell lineage distribution (p<0.002 Figure 7C). The early shift in IR isoforms suggests a potential role for IR-A in Wnt1 tumor initiation. Finally, we also measured phosphorylation and expression levels of insulin receptor substrate 1 (IRS-1) in MMTV-Wnt1 and bigenic tumors. Downregulation of IGF-1R and IRS-1 correlates with breast tumor progression, and their levels are inversely correlated with high proliferation rates and dedifferentiated breast cancers (61). Furthermore, in prostate cancer, an increase in the IR-A:IR-B ratio is positively correlated with a decrease in IRS-1 expression (62). Consistent with the increase in the IR-A:IR-B ratio in the bigenic tumors, the level of P-IRS-1 was decreased in the bigenic tumors (p=0.05 Figure 7D). Interestingly, total IRS-1 was unaltered in the bigenic tumors.

The increased ratio of IR-A:IR-B indicates that the bigenic tumors will respond differently to circulating insulin based on the mitogenic/growth properties of the IR-A isoform. However, IGF-II also binds the IR-A with high affinity, and activation of IGF-II/IR-A signaling is prevalent in a variety of cancers (18-20). Interestingly, expression of IGF-II was significantly higher in the bigenic tumors (p=0.05; Figure 7E). The increase in IGF-II expression is consistent with the increase in the CD61+ luminal progenitors, since they are known to produce IGF-II (63). IGF-II vs insulin signaling through IR-A results in distinct gene expression profiles; this supports a unique role for IGF-II through IR-A in the bigenic tumor progression (64). Finally, since both IGF-II and IR-A:IR-B expression levels were elevated in the bigenic tumors, we hypothesized that this signaling pathway may be responsible for increased canonical Wnt signaling in the bigenic tumors. Using an IGF-1R null (R-) fibroblast cell line overexpressing the IR-A (65) we found that IGF-II increased β-catenin levels in a dose dependent manner (10 nM IGF-II, p=0.18; 50 nM IGF-II, p=0.05; 100 nM IGF-II, p=0.008; Figure 7F).

These data show that reduction of IGF-1R signaling in the mammary epithelium in a MMTV-Wnt1 tumor leads to an increase in the canonical Wnt signaling target, β -catenin, and enhances the IR-A:IR-B ratio known to be elevated in many breast cancers. The stabilization of β-catenin has been shown previously to increase squamous transdifferentiation in MMTV-Wnt1 tumors (40). The increase in β-catenin in the bigenic tumors suggests that loss of IGF-1R signaling helps stabilize β-catenin levels and thus enhances the MMTV-Wnt1 tumor phenotype, driving the resulting tumors towards a more squamous and basal cell phenotype.

Conclusions

These data show that disruption and attenuation of the IGF-1R during pubertal growth of the mammary gland alters cell lineage distribution, which may also alter susceptibility to oncogenic transformation. In our model, we observed a decrease in tumor latency in the bigenic mouse model compared to MMTV-Wnt1 mice. The resulting tumors were more basal and had increased expression of EMT markers, suggesting the tumors had a more aggressive phenotype. The attenuation of the IGF-1R in the MMTV-Wnt1 tumors led to an increase in Notch targets as well as the IR-A:IR-B ratio. The increase in Notch signaling suggests that the IGF-1R may play a role either directly or indirectly in regulating Notch signaling. Taken together, the results suggest that IGF-1R and IR-A signaling have inverse roles in MMTV-Wnt1 oncogenesis and that inhibiting the IGF-1R increases signaling through the IR-A and, subsequently, enhances canonical Wnt signaling.

Precis.

Although IGF-1R inhibitors are being considered for treatment of basal-like breast cancers, the results of this preclinical study suggest that attenuating IGF-1R in the context of activated Wnt signaling in this subtype leads to negative outcomes, with possible implications for attendent clinical cautions in this direction.

Acknowledgements

This work was funded by National Institute of Diabetes, Digestive and Kidney Diseases [DK60612] to TLW, the Ruth Estrin Goldberg Foundation to DAL and NJCCR #DFHS12CRP011 Fellowship to MES, LA and LMR. The authors would like to thank Sukhwinder Singh for assistance with FACs and analyses. We would also like to thank Dr. Yi Li at the Baylor College of Medicine for helpful discussions on the MMTV-Wnt1 tumors and Dr. Andrea Morrione at Thomas Jefferson University for providing the R-/IR-A cell line.

Footnotes

The authors disclose no potential conflicts of interest.

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