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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2016 Feb 29;113(11):3030–3035. doi: 10.1073/pnas.1523693113

Pten loss promotes MAPK pathway dependency in HER2/neu breast carcinomas

Saya H Ebbesen a,b,c, Maurizio Scaltriti d,e, Carl U Bialucha b,1, Natasha Morse d, Edward R Kastenhuber c,f, Hannah Y Wen e, Lukas E Dow c,2, José Baselga d,g,h, Scott W Lowe c,i,3
PMCID: PMC4801318  PMID: 26929372

Significance

PTEN mutations are associated with disease progression and therapy resistance in human epidermal growth factor receptor 2 (HER2/neu)-amplified breast cancer patients but the role of PTEN loss in breast cancer maintenance is unknown. Here, using a regulatable RNAi mouse model of HER2/neu-driven metastatic breast cancer, we show that Pten silencing accelerates disease progression and that restoration of endogenous Pten expression triggers marked disease regression. By comparing and contrasting how pharmacologic perturbations of various signaling pathways compare to genetic reactivation of Pten, we identify a requirement for Mek signaling in Pten-suppressed tumors. Our findings imply that even advanced tumors can remain dependent on Pten loss and provide a rationale for exploring the utility of MEK inhibitors in therapy-resistant breast cancer patients acquiring PTEN mutations.

Keywords: breast cancer, mouse models, tumor suppressors, RNAi, targeted therapies

Abstract

Loss of the tumor suppressor gene PTEN is implicated in breast cancer progression and resistance to targeted therapies, and is thought to promote tumorigenesis by activating PI3K signaling. In a transgenic model of breast cancer, Pten suppression using a tetracycline-regulatable short hairpin (sh)RNA cooperates with human epidermal growth factor receptor 2 (HER2/neu), leading to aggressive and metastatic disease with elevated signaling through PI3K and, surprisingly, the mitogen-activated protein kinase (MAPK) pathway. Restoring Pten function is sufficient to down-regulate both PI3K and MAPK signaling and triggers dramatic tumor regression. Pharmacologic inhibition of MAPK signaling produces similar effects to Pten restoration, suggesting that the MAPK pathway contributes to the maintenance of advanced breast cancers harboring Pten loss.

The PTEN (phosphatase and tensin homolog) tumor suppressor gene is mutated or silenced in a wide range of tumor types (1). PTEN encodes a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase that counters the action of the phosphatidylinositol 3-kinases (PI3Ks), which otherwise transmit growth factor signals from receptor tyrosine kinases to downstream mediators such as the AKT family of serine/threonine-specific protein kinases (2). AKT, in turn, activates a series of downstream effectors that promote cellular proliferation and survival. Consequently, PTEN loss leads to hyperactivation of the PI3K pathway, and it is widely believed that this is the primary mechanism whereby PTEN loss drives tumorigenesis (3). Although cross-talk and feedback signaling makes the situation more complex (4), this molecular framework provides a strong rationale to target PI3K pathway components in PTEN-deficient tumors, and indeed, a variety of small-molecule antagonists with such activities are currently in clinical trials (5, 6).

Deregulation of the PI3K pathway is common in breast cancer and most frequently occurs through mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (7). By contrast, PTEN mutation or loss is less frequent at diagnosis but instead is associated with disease progression (8, 9). For example, PTEN inactivation often arises in oncogenic receptor tyrosine kinase human epidermal growth factor receptor 2 (HER2/neu) amplified tumors in patients who acquire resistance to the HER2/neu targeting agent trastuzumab (10–14). Similarly, PTEN mutations were recently reported in a patient harboring PIK3CA mutations that developed resistance to the PI3Kα inhibitor BYL719 (15). Thus, PTEN inactivation occurs in advanced disease in patients with poor prognosis, defining a breast cancer subtype for which there is an unmet clinical need.

Studies using mouse models have confirmed the importance of PI3K signaling in breast cancer (16). Transgenic mice that overexpress mutant PIK3CA in conjunction with HER2/neu recapitulate resistance to anti-HER2/neu therapies (17), and conditionally overexpressed mutant PIK3CA in the mammary gland gives rise to tumors at long latency that regress upon oncogene withdrawal (18). Although these observations contribute to the rationale for targeting PI3K pathway components in breast cancer, they use a model in which mutant PIK3CA is expressed at unphysiological levels and serves as the initiating event. Furthermore, studies using conditional knockout mice indicate that deregulation of the endogenous PI3Ks indirectly through Pten inactivation can promote advanced disease in combination with HER2/neu (19, 20). Still, whether sustained PTEN inactivation is needed for the maintenance of advanced cancers remains unknown. Resolving this issue may reveal cellular dependencies and, as such, instruct the clinical use of molecularly targeted agents attacking the PTEN network. In this study, we explore the impact of genetic and pharmacologic manipulation of the Pten pathway in breast cancer. Unexpectedly, our results reveal that Pten loss is required to maintain advanced disease by enhancing signaling through both the PI3K and mitogen-activated protein kinase (MAPK) cascades.

Results

A Model for Mammary Gland-Specific Pten Silencing.

Genetically engineered mouse models (GEMMs) are powerful tools for the study of gene function in disease (21, 22). We previously optimized an efficient pipeline that implements recombinase-mediated cassette exchange to introduce tet-responsive shRNAs into embryonic stem cells at a defined genomic locus, thereby providing a platform to explore the requirement for sustained gene loss in disease progression and maintenance (23, 24). To build a GEMM that enables inducible and reversible knockdown of Pten in the mammary epithelium, we bred mice carrying a whey acidic protein (WAP) gene promoter-driven Cre transgene (25) and a CAGs-LoxPStopLoxP-rtTA3-ires-mKate2 (CAGs-LSL-RIK) allele (26), which together drive mammary luminal ductal epithelium-restricted expression of a reverse tet-transactivator (rtTA3) and a far-red fluorescent protein (mKate2) in response to lactogenic hormones. The expression of rtTA3 enables doxycycline (dox)-dependent GFP-linked shRNAs (TRE-GFP-miR30-shRNA or TG-shRNA) to be expressed from a transgene integrated downstream of the col1a1 locus (Fig. 1 A–C and Fig. S1A).

Fig. 1.

Fig. 1.

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Mammary gland-specific expression of dox-inducible miR30 shRNAs. (A) Schematic description of the five transgenic alleles incorporated into the “Dox-On” multiallelic model, with the CAGs-LSL-rtTA3-IRES-mKate2 (CAGs-LSL-RIK) allele shown in its configuration before and after Cre-mediated recombination upon lactogenic stimulus. This RIK transgene contains a CMV early enhancer element and chicken beta actin (CAGs) ubiquitous promoter upstream of the LSL, followed by a modified reverse tet-transactivator with increased dox sensitivity (rtTA3), internal ribosomal entry site (IRES), and a monomeric far-red fluorescent protein (mKate2). Because the lactation-induced WAP promoter provides the tissue specificity, we opted for the use of a ubiquitous promoter upstream of the rtTA element such that its expression levels are not modulated by changes in cellular state over the course of tumorigenic transformation. Following pregnancy, the CAGs promoter drives constitutive expression of rtTA3 and fluorescent reporter mKate2. Once the luminal cells of the mammary ductal epithelium express rtTA3, dox can be used to induce expression of the shRNA transgenic allele TRE-GFP-miR30-shRNA (TG-shRNA). The tetracycline-responsive element promoter (TRE) is active when bound by rtTA3, and GFP has been coupled with the expression of the shRNA such that the strength of the fluorescent signal corresponds inversely to the knockdown level of the target protein. The resulting model provides postadolescent, luminal epithelial-specific knockdown of the target protein of interest. (B) Time line of procedural steps for transgene activation including lactation and dox treatment. Experimental animals (carrying either the Ren.713 control shRNA or Pten shRNA) are generated as littermate cohorts to control for the influence of the mixed genetic background. Mice are mated at 7 wk of age after the development of the mammary gland, and pregnancy lasts 21 d. Lactation after litter birth induces the WAP promoter, and as such dox treatment is started simultaneously to litter birth to induce shRNA expression. Nursing pups are weaned after 3 wk, and experimental mice are monitored weekly for onset of tumor growth. (C) Representative images of immunofluorescence (IF) analysis of mammary glands from parous WAP-Cre/CAGs-LSL-RIK/TG-shRen.713 (abbreviated as “shRen”) control animals either with or without dox treatment. Fluorescent reporter mKate2 staining indicates cells expressing rtTA3. In the presence of dox, rtTA3 binds the TRE promoter and drives expression of the GFP reporter and shRNA. (Scale bars: 20 μm.) (D) Representative images of IHC analysis of mammary glands from parous, dox-treated shRen and shPten (abbreviation for WAP-Cre/CAGs-LSL-RIK/TG-shPten.1522) animals. GFP staining indicates cells expressing miR30 shRNAs, and Pten protein loss is observed specifically in luminal ductal cells in shPten ducts. (Scale bars: 20 μm.)

Fig. S1.

Fig. S1.

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Model characterization and Pten knockdown in parous adult tissue. (A) Representative images of IF analysis of mammary glands from parous, dox-treated shRen animals. Fluorescent reporter mKate2 staining indicates cells expressing rtTA3. CK19 and CK8 are markers specific to the luminal cells of the mammary epithelium. (Scale bars: 20 μm.) (B) Representative optical in vivo imaging of parous, dox-treated littermates. Genotypes: WAP-Cre/CAGs-LSL-RIK/TG-shPten.1522 (Left) and WAP-Cre/TG-shPten.1522 (no rtTA3) (Right). (C) IHC staining confirming Pten target knockdown in shPten.2049 (abbreviation for WAP-Cre/CAGs-LSL-RIK/TG-shPten.2049) animals. (Scale bars: 50 μm.) (D) Analysis of mammary glands through carmine red staining of mammary epithelial whole mounts. (Scale bars: 5 mm.)

In this dual-color “Dox-On” system, mKate2 reports Cre recombinase activity and expression of rtTA3, whereas GFP fluorescence identifies cells with shRNA expression and Pten silencing (23). Noninvasive, in vivo imaging of fluorescent markers allows for detection of transgene activation and longitudinal surveillance of tumor progression (Fig. S1B). We used two transgenic shRNA strains that demonstrate efficient Pten silencing to confirm that our phenotypes were due to loss of Pten and not shRNA-specific off-target effects (27). Luminal specific expression of shRNAs targeting Pten in postpregnancy (parous) adult animals conferred robust target protein suppression (Fig. 1D and Fig. S1C) but did not result in morphological defects of the mammary glands (Fig. S1D). Both WAP-Cre/CAGs-LSL-RIK/TG-shPten.1522 (abbreviated hereafter as shPten) and WAP-Cre/CAGs-LSL-RIK/TG-shPten.2049 (shPten.2049) animals that were affected only by Pten knockdown did not develop tumors for up to 300 d of dox treatment (n > 6 for both shRNAs).

Pten Loss Accelerates HER2/neu-Driven Disease Onset and Progression.

To drive breast tumor initiation, we included a mammary-specific HER2/neu oncogene (MMTV-neuV664E) (28) (Fig. 1A). Continuous postpartum dox treatment of WAP-Cre/CAGs-LSL-RIK/MMTV-neuV664E/TG-shPten.1522 (abbreviated thereafter as shPten/neu) animals led to the onset of palpable mammary tumor development in 100% of mice at a median latency of 62 d (Fig. 2A). At this time no control animals (WAP-Cre/CAGs-LSL-RIK/MMTV-neuV664E/TG-shRenilla.713 or shRen/neu) showed palpable tumors (median latency 170 d), demonstrating a strong synergistic effect of HER2/neu overexpression and Pten depletion (Fig. 2A). As WAP-Cre induction is linked to lactation, we confirmed that disease burden was not related to the litter size of parous mice (Fig. S2A).

Fig. 2.

Fig. 2.

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Pten loss accelerates HER2/neu-driven disease onset and progression. Kaplan–Meier (KM) curves for cohorts of parous, dox-treated quadruple transgenic mice (WAP-Cre/CAGs-LSL-RIK/MMTV-neuV664E/TG-shRNA abbreviated as “shRNA/neu” with corresponding shRNA target gene name) monitored weekly through palpation showing (A) tumor-free and (B) overall survival for shPten/neu (n = 42) and shRen/neu (n = 46). Age has been normalized to date of litter birth, which was also the start of dox treatment in this experiment. (C) Representative images of IHC analysis of primary tumor tissue of parous, dox-treated shRen/neu and shPten/neu mice at end-stage disease showing robust HER2/neu overexpression in both cohorts and strong Pten protein knockdown specifically in shPten/neu tumors. (Scale bars: 100 μm.)

Fig. S2.

Fig. S2.

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Primary disease phenotype. (A) No statistical difference was observed in litter counts for parous animals in shRen/neu (n = 55) and shPten/neu (n = 56) cohorts. Not significant (NS) by two-tailed unpaired t test. (B) Statistically significant reduction in time from disease onset until disease endpoint between parous, dox-treated shRen/neu (n = 18) and shPten/neu (n = 31) cohorts. **P < 0.05 by two-tailed unpaired t test. (C) Representative whole-mount epifluorescence images of primary tumors observed in mice that achieved end-stage disease, including a higher magnification image of GFP-expressing ductal epithelium observed in mammary fat pad tissue encasing GFP-negative shRen/neu tumor. (D) Schematic of quadrant scoring system used to define loss of tumor-free survival and monitor progression that allowed for independent scoring of disease events in four regions of each animal’s mammary epithelial tissue as divided left/right and abdominal/thoracic. Percentage of parous, dox-treated shRen/neu (n = 31) and shPten/neu (n = 40) cohorts at end-stage disease with varied disease penetrance is quantified by the number of quadrants affected by palpable tumors. (E) Average total tumor burden volume per mouse at end-stage disease per cohort, weighed at harvest: shRen/neu (n = 9) and shPten/neu (n = 15). *P < 0.05 by two-tailed unpaired t test.

shPten/neu mice showed a dramatic decrease in overall survival compared with shRen/neu control animals, which eventually succumb to the oncogenic effect of the HER2/neu transgene (Fig. 2B and Fig. S2B). Although every tumor harvested from shPten/neu mice expressed the GFP and mKate2 fluorescent reporters, in shRen/neu mice only one quarter (26%) of observed tumors were GFP/mKate2-positive, indicating that Pten silencing promoted accelerated tumor growth in a WAP-Cre allele-expressing subpopulation of epithelial cells that does not overlap completely with the population of cells that express the MMTV-neuV664E allele (Fig. S2C). A significant increase in disease burden per mouse at end-stage disease was also seen in shPten/neu mice (Fig. S2 D and E). These results recapitulate previous studies using a conditional Cre-LoxP model of Pten loss (19, 20) and highlight the effectiveness of the shRNA approach.

Pten protein depletion was consistently robust in shPten/neu tumor tissue, which also expressed GFP (Fig. 2C). These areas also displayed strong staining for HER2/neu at the cell membrane and a high Ki-67 index, a marker for cellular proliferation (Fig. 2C and Fig. S3A). Pten protein was detected in all tumors analyzed from shRen/neu mice (n = 18/18; Fig. 2C), indicating that HER2/neu-driven tumor development did not select for stochastic Pten loss. Histopathology of primary mammary tumors revealed large nodular nests with central necrosis, similar in morphology to the parental MMTV-neuV664E strain (Fig. S3B). Both shRen/neu and shPten/neu tumors at end-stage disease showed characteristics of human invasive ductal carcinomas, with marked nuclear pleomorphism, high mitotic count, and lack of tubule formation. Thus, the shPten/neu mice develop tumors that reflect the most common malignant breast tumor type in humans and show pathological characteristics (high grade, poorly differentiated) that carry a poor clinical prognosis. Immunohistochemical analysis revealed estrogen receptor alpha (ERα) and CK19 expression in both control and Pten knockdown tumors (Fig. S3A). In contrast, there was a decrease in HER3/ErbB3 protein levels detected only in shPten/neu tumor tissue (Fig. S3A), which, in line with previous observations (29, 30), is suggestive of elevated PI3K/Akt activity and increased pathway feedback. Collectively, these results demonstrate strong cooperation between Pten loss and activated HER2/neu in a histopathologically accurate model of luminal epithelial breast cancer.

Fig. S3.

Fig. S3.

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Histopathology of primary mammary tumors at end-stage disease. (A) Representative images of IHC staining of primary tumor tissue of parous, dox-treated shRen/neu and shPten/neu mice. (Scale bars: 100 μm.) (B) Representative H&E histopathology of primary tumor tissue of parous, dox-treated shRen/neu and shPten/neu mice. (Scale bars: 100 μm.)

We also noted increased metastatic burden in shPten/neu animals (n = 19/21) compared with shRen/neu controls (n = 9/13) at end-stage disease (Fig. S4 A and B). Histologically, lung metastases resembled the primary tumors (Fig. S4C), showing strong HER2/neu overexpression and Pten knockdown (Fig. S4D). Importantly, key phenotypes (accelerated tumor initiation and progression, lung metastasis, and histopathology) were validated with two independent shRNAs targeting Pten (Fig. S5). Together, our results demonstrate that shRNA-mediated Pten silencing has a dramatic impact on tumor initiation and progression in HER2/neu-mediated mammary tumorigenesis.

Fig. S4.

Fig. S4.

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Metastatic phenotype. (A) Representative whole-mount epifluorescence images of spontaneous metastasis observed in parous, dox-treated shRen/neu and shPten/neu mice at end-stage disease. White arrow: GFP-negative met. (B) Quantification of lung metastasis observed in parous, dox-treated shRen/neu (n = 13) and shPten/neu (n = 21) mice at end-stage disease using as marker HER2/neu IHC staining. NS by two-tailed unpaired t test. (C) Representative H&E histopathology of metastatic lung lesions in parous, dox-treated shPten/neu mice. (Scale bars: 100 μm.) (D) Representative H&E histopathology and IHC staining of metastatic lung lesions of parous, dox-treated shPten/neu mice confirming sustained Pten knockdown, presence of GFP marker, and robust HER2/neu overexpression. (Scale bars: 100 μm.)

Fig. S5.

Fig. S5.

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Data from second shRNA strain TG-shPten.2049. KM curves comparing cohorts of parous, dox-treated quadruple transgenic shPten.1522/neu (n = 42; data presented in Fig. 2) and shPten.2049/neu (n = 23) mice monitored weekly through palpation showing (A) tumor-free and (B) overall survival. Age has been normalized to date of litter birth, which was also the start of dox treatment in this experiment. (C) Representative images of IHC analysis of primary mammary tumors from parous, dox-treated shPten.2049/neu mice. (Scale bars: 100 μm.) (D) Representative whole-mount epifluorescence images of primary mammary tumors and spontaneous lung metastasis observed in parous, dox-treated shPten.2049/neu mice at end-stage disease.

Restoration of Pten Causes Tumor Regression.

To assess whether sustained Pten loss is required for tumor maintenance, we took advantage of the unique ability of regulated RNAi to reverse gene silencing and restore endogenous Pten protein in established tumors. For this, we identified a cohort of shPten/neu animals with palpable tumors in at least one quadrant region (n = 19, 52–97 d dox treated), withdrew dox to restore Pten expression, and monitored tumor growth with caliper measurements and small animal magnetic resonance imaging (MRI). Restoration of Pten protein and loss of shRNA-linked GFP was confirmed in tumor tissue 2 wk after dox withdrawal (Fig. 3A).

Fig. 3.

Fig. 3.

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Pten loss is required for tumor maintenance. (A) Representative images of IHC analysis of hyperplastic ductal epithelium of the mammary gland or mammary tumor of parous shPten/neu mice either continuously dox treated or 2 wk after dox withdrawal. (Scale bars: 100 μm.) (B) Representative axial plane MRI images of continuously dox-treated (n = 2) and dox-withdrawn (n = 3) cohorts of shPten/neu mice analyzed by MRI at days 0 and 14. (C) Waterfall plot displaying percentage of change in total tumor burden volume per mouse at the time point of 3 wk after dox withdrawal for a cohort of shPten/neu mice (n = 17) compared with a representative cohort of continuously dox-treated animals (n = 8). Dotted lines indicate +100 and −30% thresholds. (D) KM curve depicting the overall survival benefit of animals after dox withdrawal (n = 19) compared with dox-treated animals (n = 56; data partially presented in Fig. 2). Double arrow indicates range of dox withdrawal start date.

Most tumors arising in shPten/neu mice showed dramatic, and often complete, regression during the first 3 wk following dox withdrawal (n = 15/17), whereas those in shPten/neu animals maintained on dox exhibited exponential tumor growth (Fig. 3 B and C). Tumor regression was indeed due to Pten protein restoration, as tumors in the second shRNA strain, shPten.2049/neu, displayed similar regression patterns whereas those arising in shRen/neu control mice at long latency remained unaffected by dox treatment withdrawal (Fig. S6). Neither total tumor burden at the time of dox withdrawal nor duration of dox treatment before dox withdrawal correlated with the maximal response in tumor regression documented in each animal (Fig. S7). In most cases, tumor regression was sustained for at least 50 d after dox withdrawal (Fig. S8), leading to a significant increase in overall survival (Fig. 3D). Nonetheless, multifocal disease relapse driven by HER2/neu overexpression eventually occurred in almost all shPten/neu mice maintained off dox (n = 18/19) (Fig. 3D). Still, this dramatic regression of exponentially growing HER2/neu-driven mammary tumors upon Pten restoration suggests that Pten knockdown not only accelerates tumor initiation but also is required for tumor maintenance.

Fig. S6.

Fig. S6.

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Tumor regression data: controls. (A) Waterfall plot displaying maximal percentage of change in total tumor burden volume per mouse relative to day 0 during first 35 d of dox withdrawal for cohorts of shRen/neu (n = 5) and shPten.2049/neu (n = 4) mice, compared with a representative cohort of continuously dox-treated shRen/neu animals (n = 4). Dotted lines indicate +100 and −30% thresholds. (B) Representative axial plane MRI images of a dox-withdrawn cohort of shPten.2049/neu mice monitored by MRI (n = 3) at days 0 and 20.

Fig. S7.

Fig. S7.

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Maximal response correlation statistics. (A) Maximal response per mouse (n = 17) recorded in total tumor burden after dox withdrawal relative to day of withdrawal (T0), plotted against total tumor burden per animal at T0. Lack of correlation (r = 0.1074) demonstrates no significant impact of initial tumor burden on degree of tumor regression. NS by two-tailed P value for Pearson’s r coefficient. (B) Maximal response per mouse (n = 19) recorded in total tumor burden after dox withdrawal relative to day of withdrawal (T0), plotted against duration of dox treatment before dox withdrawal. Lack of correlation (r = −0.2503) demonstrates no significant impact of dox-treatment duration before withdrawal on degree of tumor regression. NS by two-tailed P value for Pearson’s r coefficient.

Fig. S8.

Fig. S8.

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Total tumor burden volume graphs. (A) Total tumor burden volume per mouse plotted against time since dox withdrawal for a cohort of dox-withdrawn animals (n = 17) compared with a representative control cohort of continuously dox-treated animals (n = 8). (B) Data represented as relative change over time in total tumor burden volume relative to burden at T0.

Pten Loss Drives PI3K/Akt and MAPK Signaling Pathways in HER2/neu Mammary Tumors.

We postulated that restoration of Pten protein in Pten knockdown tumor tissue disrupts the signaling pathways governing cellular proliferation and/or survival, leading to tumor shrinkage. To explore this in more detail, we performed immunohistochemical (IHC) and immunoblot (IB) analyses of shRen/neu and shPten/neu primary mammary tumors (Fig. 4 and Fig. S9A). In regressing shPten/neu tumors examined 3 wk after dox withdrawal, tumors showed reduced proliferation (as measured by BrdU incorporation and Ki-67 staining) but no detectable increase in apoptosis (as assessed by cleaved caspase-3 staining) (Fig. S9B). Although these observations suggest that tumor shrinkage was independent of apoptosis, we cannot rule out the possibility that transient increases in signal were missed at the examined time point. Consistent with the ability of Pten to suppress PI3K signaling, Pten knockdown tumors showed an increase in Akt phosphorylation that was not apparent in tumors arising in shRen/neu mice (Fig. 4B). Akt levels were suppressed upon Pten restoration, and notably, relapsed tumors maintained re-expression of Pten (Fig. 4).

Fig. 4.

Fig. 4.

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Heightened PI3K/Akt and MAPK pathway signaling in Pten knockdown tumors. IHC (A) and IB (B) analysis of end-stage disease mammary tumors of parous shRen/neu and shPten/neu mice either continuously dox treated or dox withdrawn. (Scale bars: 100 μm.) For B, unsorted whole tumor protein lysate was analyzed.

Fig. S9.

Fig. S9.

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Additional IHC analysis and lung data. (A) Representative images of additional IHC analysis of end-stage disease mammary tumors of parous shRen/neu and shPten/neu mice, either continuously dox treated or dox withdrawn. (Scale bars: 100 μm.) (B) IHC analysis of mammary tumors post-BrdU pulse in parous shPten/neu mice, either continuously dox treated or 3 wk after dox withdrawal. (Scale bars: 200 μm.) (C) IHC analysis of spontaneous lung metastatic lesions of parous, dox-treated shRen/neu and shPten/neu mice with end-stage disease. (Scale bars: 200 μm.) (D) IHC analysis of mammary tumors from shPten/neu mice 7 d after dox withdrawal. (Scale bars: 100 μm.)

Unexpectedly, we also noted a consistent elevation in phosphorylated Erk (p-Erk) levels in primary tumors and pulmonary metastases arising in dox-treated shPten/neu mice compared with shRen/neu mice, suggesting that Pten suppression triggered hyperactivation of the MAPK pathway (Fig. 4 and Fig. S9C). Indeed, high p-Erk expression was not merely a secondary event in tumors generated by Pten knockdown, as restoration of endogenous Pten expression in shPten/neu tumors led to a reproducible suppression of p-Erk levels (Fig. 4). Consistent with the delay seen in the shPten/neu cohort tumor regression response, Pten levels remained low at 7 d after dox withdrawal (Fig. S9D) but returned to baseline by 14 d (Fig. 4B). High p-Akt and p-Erk levels were sustained at 7 d post-dox withdrawal but were suppressed at 2 wk. Thus, despite constitutive overexpression of an overactive HER2/neu oncogene, restoration of endogenous Pten induced a striking reduction in both PI3K/Akt and Mek/Erk signaling that correlated with disease regression.

Confounding further in-depth analysis of the relapsed tumors is the multifocal nature of the disease that develops in this GEMM. Despite our capacity to track mKate2-expressing cells in the absence of dox, given the large number of tumor-initiating events documented per mouse, it was difficult to determine whether the tumors that progressed off dox were arising from relapses of an originally responsive malignancy or rather from “background” tumors expected to arise eventually from HER2/neu-expressing cells that never expressed the Pten shRNA. Indeed, it seems most likely that they represented a mixture of both classes, as some expressed high levels of the lineage tracing mKate2 reporter linked to the rtTA3 (indicating they can express the Pten shRNA when dox treated) whereas others did not. Regardless, all of the tumors that progressed showed abundant Pten expression and did not contain elevated p-Akt or p-Erk levels, indicating that secondary mechanisms capable of recapitulating Pten loss were not acquired.

Mek Signaling Is Required to Maintain Tumors with Suppressed Pten.

The marked reduction in PI3K/Akt and Mek/Erk signaling following Pten restoration prompted us to ask whether the activity of each pathway was critical for disease progression and, thus, whether pharmacological inhibition could also drive tumor regression. To provide a controlled experimental setting for comparing different treatment arms, we generated cohorts of matched secondary transplants from primary tumor fragments harvested from shPten/neu mice. These transplants showed consistent tumor growth (on dox) and response to Pten restoration (dox withdrawn), mirroring the primary disease in transgenic mice (Fig. 5). In all, we assessed four treatment arms using agents targeting oncogenic HER2/neu (Lapatinib), PI3K/Akt signaling (α-Akt: MK-2206; α-pan-PI3K: NVP-BKM120), or Mek/Erk activity (α-Mek: GSK1120212) at doses routinely used in preclinical studies (15, 29, 31, 32). Drug efficacy against the mouse protein target was verified by assessing the impact on signaling in treated tumor protein extracts (Fig. S10).

Fig. 5.

Fig. 5.

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Pharmacologic inhibition of MAPK pathway recapitulates Pten restoration. (A) Dox-treated shPten/neu animal-derived primary tumor fragment serial transplantation assay in nude mice. Mice were treated with or without dox with a drug or vehicle (n = 12–14 tumors). Drug dosages: GSK1120212 (3 mg/kg daily), MK-2206 (360 mg/kg daily and then twice weekly after day 14), NVP-BKM120 (31.35 mg/kg daily), and Lapatinib (100 mg/kg daily). Data represent mean ± SEM. (B) Representative mKate2 and GFP fluorescence images from in vivo Xenogen IVIS Spectrum imaging of dox-treated and dox-withdrawn (day 10) nude mice from the tumor fragment transplantation assay.

Fig. S10.

Fig. S10.

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Inhibitor pharmacodynamics. IB analysis of tumor tissue in dox-treated shPten/neu animal-derived primary tumor fragment serial transplantation assay in nude mice (A) for inhibitor pharmacodynamics of GSK1120212, MK-2206, and NVP-BKM120 (tumors were harvested 4 h after the third daily treatment); (B) for inhibitor pharmacodynamics of Lapatinib (tumors were harvested 4 h after the third daily treatment); and (C) for efficacy of shRNA expression reversal in dox-withdrawn tumor samples (tumors were harvested 11 d after dox withdrawal).

Consistent with the known role for PI3K pathway activation in promoting resistance to HER2/neu inhibition (33, 34), Lapatinib had only a mild effect in slowing tumor growth (Fig. 5A). By contrast, pan-PI3K and Akt inhibitors, as predicted, were effective in suppressing tumor growth, although not nearly as efficiently as Pten restoration. Surprisingly, Mek inhibition induced the most robust tumor response, effectively blocking tumor growth over 4 wk of treatment and phenocopying the effect of Pten restoration in these transplants (Fig. 5A). Although counterintuitive to the canonical view of Pten as a regulator of PI3K signaling, our results suggest that Pten loss, in addition to up-regulating the PI3K/Akt pathway, causes a functional “cross-activation” of the MAPK pathway that goes beyond the canonical HER2/neu-dependent signaling through Erk and contributes significantly to aggressive tumor growth. Still, it remains a possibility that pan-PI3K and Akt inhibition was less effective than Mek inhibition in our model due to differences in pharmacokinetics, reduced activity against the mouse protein(s), or PI3K-independent functions of Pten (35).

Discussion

Our findings are consistent with previous work in which withdrawal of a conditional PIK3CA transgene used to initiate tumorigenesis also produced marked disease regression (18). In our study, Pten inactivation served as a cooperating event to promote tumorigenesis together with the strong initiating driver HER2/neu. Even so, Pten reactivation blunted PI3K and MAPK signaling, leading to a potent antitumor response. Although these studies suggest that pharmacological strategies to achieve similar ends would be therapeutically beneficial in advanced forms of breast cancer, the relative contribution of Pten loss to tumor maintenance may depend on tissue and/or genetic context. Accordingly, we recently showed that the antitumor effects of Pten reactivation in a mouse model of aggressive leukemia were largely limited to leukemic cells at disseminated sites (27).

Additional studies will be required to fully elucidate the complex cross-talk between the PI3K and MAPK pathways (4). Physiologically, PTEN-dependent early endodermal morphogenesis seems to require the Erk, but not the Akt, pathway (36). Furthermore, in Ras-induced oncogenic transformation, PTEN apoptotic function is suppressed via the Raf-Mek-Erk pathway (37). Recent publications using human breast cancer cell lines support the notion that PI3K inhibition can involve an ERK-dependent component (38, 39). In one study, this effect was apparently mediated by the phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1), which activates Rac1, leading to MAPK pathway activation. In another, enforced expression of PTEN caused a decrease in p-ERK levels. Both confirm that combined inhibition of MEK in conjunction with PI3K or AKT improved antitumor efficacy in breast cancer xenografts. These data are in agreement with earlier reports showing that expression of functionally active PTEN inhibits ERK activation in glioblastoma models (40). Moreover, ectopic expression of PTEN in MCF7 cells results in both AKT and ERK suppression, but only specific inhibition of MEK can abolish the negative effects of PTEN on insulin-mediated cell growth (41).

Our finding that shPten/neu primary tumor regression upon restoration of Pten protein levels is dependent on both PI3K/Akt and MAPK pathways reiterates the presence of a complex and interconnected signaling cross-talk network that regulates these tumors’ sustained survival and proliferative advantage in a relevant physiological context. Collectively, these data provide a strong rationale for combining agents that target the MAPK and PI3K pathways, especially in the absence of PTEN (42). As PTEN loss is linked to resistance against anti-HER2/neu agents or single PI3K inhibitors, such combinatorial approaches would address an unmet clinical need.

Materials and Methods

Mouse Strains, Animal Husbandry, and Tumor Cohorts.

All mouse strains have been previously described. MMTV-neuV664E [strain name: FVB-Tg(MMTV-Erbb2)NK1Mul/J] (28) and WAP-Cre [strain name: B6.Cg-Tg(Wap-cre)11738Mam/JKnwJ] (25) were purchased from Jackson Laboratory. In the MMTV-neuV664E allele, an activating point mutation (V664E) in the transmembrane domain of neu results in increased receptor homodimerization and constitutive activation of the kinase domain (43). The strains CAGs-LSL-RIK (26), TG-shRenilla-Luciferase.713 (23), TG-shPten.1522 (27), and TG-shPten.2049 (27) were made by the S.W.L. laboratory. Transgenic strains were not backcrossed onto the same strain background before mating; thus all experimental animals were generated on a mixed strain background. The mating strategy was conducted such that the experimental shRNA and the control shRNA animals were born as littermates. Mice were housed in vented cages with a 12-h light cycle and food and water ad libitum. Experimental female animals, after PCR verification for the correct genotypes, were bred at 7 wk, and doxycycline feed was administered from the date of litter birth. Litters were nursed for 3 wk to induce WAP promoter activity. Parous mice underwent only one pregnancy cycle. Parous mice were monitored weekly, blinded from genotypic information, for tumor formation by physical palpation. Tumor and organ tissue samples were excised from humanely euthanized mice and either fixed and/or snap-frozen. Caliper measurements during tumor regression experiments were used to estimate tumor volume with the formula V = (4/3) * (pi) * (L/2) * (W/2)^2, where Length > Width. Total tumor burden volume per mouse was calculated as the sum of all tumor volumes per mouse at each particular time point.

Study Approval.

All mice were maintained and experiments were conducted as approved by the Institutional Animal Care and Use Committee (IACUC) at Memorial Sloan Kettering Cancer Center (MSKCC) under protocol nos. 11–06-015 and 12–10-019.

SI Materials and Methods

Serial Transplantation Assays.

The primary transgenic animal-derived mammary tumor fragment serial transplantation assays were conducted using 7- to 9-wk-old nude female mice purchased from Harlan Laboratories. Mammary tumors were initially harvested from a dox-treated shPten.1522/neu animal dissected into ∼1-mm3 pieces and cryopreserved. Defrosted fragments were s.c. implanted into dox-treated recipient nude mice. Tumors that developed were harvested, fragmented, and directly implanted into a secondary recipient cohort that was used for the drug treatment studies. Each mouse was implanted on both left and right flanks. Mice were randomized 26 d postimplantation for the start of inhibitor treatments. Tumor transplant dimensions were measured by caliper every 2–3 d, and tumor volume was determined using the following formula: (Length*Width^2)*(3.14159/6), where Length > Width. At the end of the study, mice were anesthetized with 1.5% isofluorane-air mixture and killed by cervical dislocation before tumor sample harvest. Tumors were removed 4 h following the last drug administration.

Treatments.

Doxycycline hyclate was administered at a dose of 625 mg/kg in alfalfa-free rodent diet with 18.2% protein, 48% carbohydrate, and 5.8% fat content (Harlan Laboratories). The diet is designed to deliver a daily dose of 2–3 mg of doxycycline based on consumption of 4–5 g/day by a mouse; doxycycline hyclate contains ∼87% doxycycline. BrdU was administered by i.p. injection 2 h before harvest at a dose of 1 mg per mouse. For pharmacological inhibition studies, mice were treated by oral gavage with inhibitors against MEK, AKT, pan-PI3K, or HER2/EGFR at the indicated doses: MEK inhibitor GSK1120212 (Trametinib)—3 mg/kg daily in 10% (vol/vol) Cremophor EL and 10% (vol/vol) PEG 400; AKT inhibitor MK-2206—360 mg/kg daily and then twice weekly after day 14 in 30% (wt/vol) Captisol; pan-PI3K inhibitor NVP-BKM120—31.35 mg/kg daily in 10% (vol/vol) NMP and 90% (wt/vol) PEG300; and HER2/EGFR dual-kinase inhibitor Lapatinib—100 mg/kg in 0.5% hydroxypropylmethycellulose, 0.1% Tween 80.

Small Animal Imaging.

Noninvasive, optical in vivo imaging was conducted using a Xenogen IVIS Spectrum. The IVIS Imaging System was prepared for use according to the IACUC Guidelines for Animal Use at MSKCC. The Small Animal Imaging Core Facility at MSKCC performed MRI according to the IACUC Guidelines for Animal Use.

Carmine Staining.

Whole-mount mammary gland carmine staining was performed using standard methods. Briefly, number 4 mammary glands were dissected out and fixed in Carnoy’s fixative (6 parts 100% ethanol:3 parts chloroform:1 part Glacial acetic acid) for 2–4 h at room temperature. After rehydration, glands were stained in carmine alum overnight at room temperature. After dehydration, samples were stored in methyl salicylate.

Histology, Immunohistochemistry, and Immunofluorescence.

Tissue was fixed in 10% (vol/vol) neutral buffered formalin overnight at room temperature. Paraffin embedding and sectioning was performed by IDEXX RADIL. Hematoxylin and eosin staining was performed using standard methods. Sections were rehydrated and unmasked (antigen retrieval) by heat treatment for 10 min in a pressure cooker in 10 mM Tris and 1 mM EDTA buffer (pH 9) containing 0.05% Tween 20. For immunohistochemistry, sections were treated with 3% (vol/vol) H2O2 for 10 min and blocked in PBS containing 5% (wt/vol) BSA. For immunofluorescence, sections were not treated with peroxidase. Primary antibodies, incubated at 4 °C overnight in blocking buffer, were the following: rabbit anti-GFP (for IHC; 1:200, Cell Signaling 2956); rabbit anti-tRFP (for mKate2; 1:5,000 for IHC, 1:1,000 for IF, Evrogen AB232 and AB234); rabbit anti-PTEN (1:100, Cell Signaling 9559), rabbit anti-ErbB2 (for HER2/neu and ErbB2; 1:500, Abcam 2428); rabbit anti-Ki-67 (1:100, Abcam 16667); rabbit anti-phospho-p44/42 MAPK (1:400, Cell Signaling 4376); rabbit anti-phospho-Akt Ser473 (1:50, Cell Signaling 4060); rabbit anti-phospho-S6 ribosomal protein S235/236 (1:200, Cell Signaling 4858); rabbit anti-CK19 (for IHC; 1:100, Abcam 15463); rabbit anti-cleaved caspase-3 (1:400, Cell Signaling 9664); rabbit anti-HER3/ErbB3 (1:250, Cell Signaling 12708); rabbit anti-ERα (1:100, Santa Cruz 542); rat anti-BrdU (1:250, Abcam 6326); chicken anti-GFP (for IF; 1:500, Abcam 13970); rabbit anti-CK19 (for IF; 1:1,000, Epitomics 3863–1); and rat anti-CK8 (1:200, DSHB “TROMA-1-s”). For immunohistochemistry, sections were incubated with anti-rabbit ImmPRESS reagent (Vector Laboratories, #MP7401) and developed using ImmPACT DAB (Vector Laboratories, #SK4105) according to the manufacturer’s instructions. For immunofluorescent stains, secondary antibodies were applied for 1 h at room temperature in Tris-buffered saline (TBS) in the dark, washed twice with TBS, counterstained for 5 min with DAPI, and mounted in ProLong Gold (Life Technologies, #P36930). Secondary antibodies used were the following: anti-rabbit 568 (1:500, Molecular Probes #a11036), anti-chicken 647 (1:500, Molecular Probes #121449), and anti-rat 568 (1:500, Molecular Probes #a11977). Images of IHC-stained sections were acquired on a Zeiss Axioscope Imager Z.1. Images of IF-stained sections were acquired on a confocal microscope at the Molecular Cytology Core Facility at MSKCC. Raw .tif files were processed using Photoshop CS5 software (Adobe Systems Inc.) to adjust levels and/or merge images. Individual tumors, individual mammary fat pads, and whole lungs were considered as distinct biological replicates. A minimum of n = 2 mammary fat pads and n = 3 tumors were probed per genotype and treatment condition for each antibody staining.

Immunoblotting.

Western blots were performed using standard methods. Briefly, protein lysates were prepared from crushed tumor tissue in Laemmli buffer supplemented with protease and phosphatase inhibitors and quantified by Lowry assay (Bio-Rad Laboratories). Whole-cell extracts were separated on 8% to 12% SDS/PAGE gels and transferred to PVDF membranes (EMD Millipore). Membranes were blocked with 5% (wt/vol) milk in TBS with 0.1% Tween 20 and probed with specific antibodies (see below). Blots were incubated with HRP-linked secondary antibodies and developed with chemiluminescence. Western blots were probed with antibodies against the following: rabbit anti-PTEN (1:1,000, Cell Signaling 9559), rabbit anti-phospho-AKT Ser473 (1:1,000, Cell Signaling 4060), rabbit anti-phospho-AKT Thr308 (1:1,000, Cell Signaling 2965), rabbit anti-AKT (1:1,000, Cell Signaling 9272), rabbit anti-phospho-p44/42 MAPK (for p-Erk; 1:400, Cell Signaling 4376), mouse anti-p44/42 MAPK (for Erk1/2; 1:2,000, Cell Signaling 9107), rabbit anti-GFP (1:1,000, Cell Signaling 2956), rabbit anti-phospho-S6 ribosomal protein S235/236 (1:2,000, Cell Signaling 4858), mouse anti-S6 ribosomal protein (1:500, Cell Signaling 2317), mouse anti-β-Actin-HRP (1:5,000, monoclonal AC15 clone, Sigma #A3854), and rabbit anti-phospho-MEK1/2 S217/221 (1:1,000, Cell Signaling 9154).

Statistics.

Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc.). The Log-rank Mantel–Cox test and Mantel–Haenszel hazard ratio were used to analyze survival data, Pearson’s r coefficient for correlations, and the two-tailed unpaired t test for all other experimental data.

Acknowledgments

We gratefully thank Drs. Sarat Chandarlapaty, Cornelius Miething, Thomas Kitzing, John P. Morris IV, Amaia Lujambio, Justin R. Cross, Leila Akkari, and Pau Castel for valuable discussion and/or critical review of the manuscript; members of the S.W.L. laboratory for helpful comments; Sha Tian, Erika Antinis-Giannetta, Danielle Grace, Meredith Taylor, and Janelle Simon for technical assistance with mouse husbandry; and members of the Small Animal Imaging Core at Memorial Sloan Kettering Cancer Center (MSKCC) for their high-quality service and consultation. This study was supported by a scholarship from the Star Centennial Foundation (to S.H.E.); a National Institutes of Health Ruth L. Kirschstein National Research Service Award predoctoral traineeship (to S.H.E. through the Watson School of Biological Sciences); National Cancer Institute (NCI) Grants R01-CA195787, U01-CA168409, and P01-CA094060 (to S.W.L.); and MSKCC Cancer Center Support Grant P30-CA008748. This work was also funded by Stand Up To Cancer Dream Team Translational Cancer Research Grant SU2C-AACR-DT0209 from the Entertainment Industry Foundation (to J.B.); the Breast Cancer Research Foundation (J.B.); and NCI Grant R03-CA187094 (to M.S.). S.W.L. is an investigator in the Howard Hughes Medical Institute and Chair of the Geoffrey Beene Cancer Research Center.

Footnotes

Conflict of interest statement: L.E.D. and S.W.L. are members of the Scientific Advisory Board and hold equity in Mirimus Inc., a company that has licensed some of the technology reported in this paper. The involvement of L.E.D. and S.W.L. in Mirimus, Inc. does not alter their adherence to PNAS’s policies on sharing data and materials. J.B. has consulted for Novartis Pharmaceuticals and is a past member of the scientific advisory board of Seragon.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523693113/-/DCSupplemental.

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