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HER2 overexpression drives Akt signaling and cell survival and HER2-enriched breast tumors have a poor outcome when Akt is upregulated. Akt is activated by phosphorylation at T308 via PI3K and S473 via mTORC2. The importance of PI3K-activated Akt signaling is well documented in HER2-amplified breast cancer models, but the significance of mTORC2-activated Akt signaling in this setting remains uncertain. We report here that the mTORC2 obligate cofactor Rictor is enriched in HER2-amplified samples, correlating with increased phosphorylation at S473 on Akt. In invasive breast cancer specimens, Rictor expression was upregulated significantly compared with nonmalignant tissues. In a HER2/Neu mouse model of breast cancer, genetic ablation of Rictor decreased cell survival and phosphorylation at S473 on Akt, delaying tumor latency, penetrance, and burden. In HER2-amplified cells, exposure to an mTORC1/2 dual kinase inhibitor decreased Akt-dependent cell survival, including in cells resistant to lapatinib, where cytotoxicity could be restored. We replicated these findings by silencing Rictor in breast cancer cell lines, but not silencing the mTORC1 cofactor Raptor (RPTOR). Taken together, our findings establish that Rictor/mTORC2 signaling drives Akt-dependent tumor progression in HER2-amplified breast cancers, rationalizing clinical investigation of dual mTORC1/2 kinase inhibitors and developing mTORC2-specific inhibitors for use in this setting. Cancer Res; 76(16); 4752–64. ©2016 AACR.

This file contains the supplemental figure legends

This file contains supplemental materials and methods

Supplemental Figure Legends and Supplemental tables (Table 1 and table 2)

This Supplemental Figure 1 describes engineering of FIP1C inducible mouse model and condition knockout. Moreover, Supplemental Figure 1 provides data related to the elucidation of the physiological role of FIP1C during mamamry gland outgrowth as a supplement of Figure 1. Overall,the modulation of FIP1C levels in mammary gland does not affect normal mammary gland development.

Supplementary methods

The invasive and metastatic properties of many human tumors have been associated with upregulation of the miRNA miR-10b, but its functional contributions in this setting have not been fully unraveled. Here, we report the generation of miR-10b–deficient mice, in which miR-10b is shown to be largely dispensable for normal development but critical to tumorigenesis. Loss of miR-10b delays oncogene-induced mammary tumorigenesis and suppresses epithelial–mesenchymal transition, intravasation, and metastasis in a mouse model of metastatic breast cancer. Among the target genes of miR-10b, the tumor suppressor genes Tbx5 and Pten and the metastasis suppressor gene Hoxd10 are significantly upregulated by miR-10b deletion. Mechanistically, miR-10b promotes breast cancer cell proliferation, migration, and invasion through inhibition of the expression of the transcription factor TBX5, leading to repression of the tumor suppressor genes DYRK1A and PTEN. In clinical specimens of breast cancer, the expression of TBX5, HOXD10, and DYRK1A correlates with relapse-free survival and overall survival outcomes in patients. Our results establish miR-10b as an oncomiR that drives metastasis, termed a metastamiR, and define the set of critical tumor suppressor mechanisms it overcomes to drive breast cancer progression. Cancer Res; 76(21); 6424–35. ©2016 AACR.

Additional details related to the Materials and Methods section that appears in the main text.

Supplemental Figure S1. Kaplan meier analysis and oncoprint mapping of Luminal A/B (A) and Basal-like (B) TCGA-curated breast cancers, showing the tumors in each set with upregulation of Akt1/2/3 mRNA (red boxes) or phospho-S473 (black arrows), and the survival of these patients (red Kaplan-Meier line) versus other patients (blue Kaplan-Meier line). Log-rank test shows insignificance. Supplemental Figure S2. Rictor expression in human breast cancer. Supplemental Figure S3. Oncoprint of TCGA-curated invasive breast cancers (TCGA, Cell 2015, N = 971) assessed for RICTOR genomic alterations. Analysis shows 8% of cases (76/971) with gene amplification, overexpression, missense mutation, or deep deletion. Supplemental Figure S4. Rictor mRNA expression was measured by qRT-PCR of total RNA from mammary glands harvested from virgin female 12-week old mice. Supplemental Figure S5. Genetic ablation of Rictor decreases growth and survival of HER2-positive breast cancer cells. Supplemental Figure S6. A. Cells were cultured 6 hours with FITC-labeled Annexin V, then imaged. B. MCF10-HER2 cells (parental, Rictor, SFN, and Rictor ZFN + exogenous expression of myc-tagged Rictor) were cultured 6 h with An-nexin V-FITC. C. The percentage of cells in panel B that were labeled with Annexin V-FITC was quantitated. Averages (midlines) ± S.D. were assessed for significance using Student€™s two-tailed unpaired T-test. Supplemental Figure S7. Rictor-mediated Akt activity is necessary and sufficient for breast cancer cell survival. Supplemental Figure S8. Cells were treated with lapatinib for the time course indicated. Total cell RNA was used for qRT-PCR to measure relative RPTOR mRNA expression. Supplemental Figure S9. Cells were cultured 7 days in 10% serum plus lapatinib (1 μM) or PP242 (1 μM) or the combination of lapatinib + PP242. Cells were stained with crystal violet. Crystal violet fluorescence was scanned using the odyssey. Representative images are shown. Supplemental Figure S10. Loss of Rictor/mTORC2 sensitizes parental and resistant cells to lapatinib in vitro Supplemental Figure S11. Cells were treated 6h with PP242 (1μM) ± lapatinib (1μM) and assessed by luminescent caspase-3/7 assay.

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Supplementary Figure 3 from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

PDF file - 193K, Bcl3 deficiency has no effect on pre-neoplastic tissue homeostasis

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PDF file - 206K, No significant changes in the number of tumor infiltrating T cells (including CD8+ and CD4+ ) upon the addition of anti-CTLA-4 mAb to HER2/Neu antibody and triciribine combination treatment

Supplementary Methods and Figure Legends

Supplementary Figure Legends 1-8 from Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis

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Supplementary Figure 3 from Akt1 and Akt2 Play Distinct Roles in the Initiation and Metastatic Phases of Mammary Tumor Progression

PDF file - 1165K, Characterization of 14-3-3zeta Transgenic Mice and Mammary Tumors (S1); Analysis of Metastasis in Mammary Tumors (S2); Apoptosis Assays in Transgenic Mice and Cell Lines (S3); IHe Staining of Ki-67 and p21 Expression (S4); p27-Targeting miRNA Expression in Human Breast Cancer Cell Lines (S5); Analysis of Signaling Pathways Involved in 14-3-3zeta-Mediated miR-221 Transcriptional Upregulation (S6); Logistic regression model (S7).

Supplementary Table 1 from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

PDF file - 25K, HER2/Neu antibody 7.16.4 and Akt inhibitor TCN combination treatment reduced tumor multiplicity and lung metastasis in PTEN-/-/NIC mice

Supplemental Figure 1: Confirmation of specific Stat3 ablation in the epithelium.

Supplementary Figure 1 from Effect of Conditional Knockout of the Type II TGF-β Receptor Gene in Mammary Epithelia on Mammary Gland Development and Polyomavirus Middle T Antigen Induced Tumor Formation and Metastasis

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Supplemental Figure 3:Stat3flx/flx/MTB/MIC tumors are unable to colonize the lungs in the presence or absence of an innate immune system

Supplementary Figure Legends 1-5 from Akt1 and Akt2 Play Distinct Roles in the Initiation and Metastatic Phases of Mammary Tumor Progression

Supplementary Figure R from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

The phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway is often dysregulated in cancer. Our previous studies have shown that coexpression of activated Akt1 with activated ErbB2 or polyoma virus middle T antigen uncoupled from the PI3K pathway (PyVmT Y315/322F) accelerates mammary tumor development but cannot rescue the metastatic phenotype associated with these models. Here, we report the generation of transgenic mice expressing activated Akt2 in the mammary epithelium. Like the mouse mammary tumor virus-Akt1 strain, mammary-specific expression of Akt2 delayed mammary gland involution. However, in contrast to Akt1, coexpression of Akt2 with activated ErbB2 or PyVmT Y315/322F in the mammary glands of transgenic mice did not affect the latency of tumor development. Strikingly, Akt2 coexpresssion markedly increased the incidence of pulmonary metastases in both tumor models, demonstrating a unique role in tumor progression. Together, these observations argue that these highly conserved kinases have distinct biological and biochemical outputs that play opposing roles in mammary tumor induction and metastasis. [Cancer Res 2009;69(12):5057–64]

PDF file - 4MB, Loss of ErbB3 impairs hyperplasia of MMTV-Neu mammary glands.

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PDF file - 31K, SacI- and SpeI/SphI-digested genomic DNA from mice with the indicated genotypes

PDF file - 209K, Proliferative capacity and apoptotic status of ErbB3delta85/delta85/NDL2-5 mammary tumors

PDF file - 269K, Primary tumour morphology and cell turnover kinetics are unaltered by Bcl3 deficiency

Supplementary Figure 4 from Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis

PDF file - 410K, Histological and immunohistochemical analysis of tumors derived from PTEN-/-/ErbB2KI mice receiving single or combination treatment

PDF file - 31K, Migration of MG1361 cells in boyden chamber assays correlated with Bcl3 mRNA levels

PDF file - 153K, Inhibition of b-catenin signalling impacts on proliferative and transcriptional properties of basal breast cancer line.

PDF file - 137K, Depletion of CD8+ cells, CD4+ cells or both during HER2/Neu antibody and triciribine combination treatment

Supplementary Table 1 from Akt1 and Akt2 Play Distinct Roles in the Initiation and Metastatic Phases of Mammary Tumor Progression

Supplementary Table 1 from Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis

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Supplementary Table 2 from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

Supplementary Figure Legends 1-3 from Activation of Murine Double Minute 2 by Akt in Mammary Epithelium Delays Mammary Involution and Accelerates Mammary Tumorigenesis

PDF file - 139K, ErbB2KI mammary tumors express basal/myoepithelial markers and contain cells co-expressing Krt8 and Krt14.

Supplementary Figure Legends 1- 4 from Overexpression of the Protein Tyrosine Phosphatase PRL-2 Correlates with Breast Tumor Formation and Progression

PDF file - 118K, Bcl3 expression is elevated in ErbB2 overexpressing human breast cancer cell lines

Supplementary Figures 1-9 from Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

PDF file - 135K, Loss of endogenous β-catenin in ErbB2KI-derived mammary tumor cells (TM15 c10-2 and c7-2) impairs their invasion capacity in vitro.

Supplementary Figure 3 from Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis