Two distinct mTORC2-dependent pathways converge on Rac1 to drive breast cancer metastasis
The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear.
Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays.
We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially increased Rac1 activity through the Rac-GEF Tiam1, thus partially rescuing cell invasion/motility. The mTORC2 effector protein kinase C (PKC)α did rescue Rictor-mediated RhoGDI2 downregulation, partially rescuing Rac-guanosine triphosphate (GTP) and migration/motility.
These findings suggest that mTORC2 uses two coordinated pathways to activate cell invasion/motility, both of which converge on Rac1. Akt signaling activates Rac1 through the Rac-GEF Tiam1, while PKC signaling dampens expression of the endogenous Rac1 inhibitor, RhoGDI2.
KeywordsmTOR Rictor Akt Rac Metastasis Breast cancer RhoGDI2 Protein kinase C HER2 Mouse mammary tumor Conditional knockout
Analysis of variance
American Type Culture Collection
Dulbecco’s modified Eagle’s medium
Epidermal growth factor
Epithelial mesenchymal transition
Guanine nucleotide exchange factors
Glutathione-S transferase-Pak binding domain
Hematoxylin and eosin
Human epidermal growth factor receptor 2
Internal ribosomal entry site
Mouse embryonic fibroblast
Neu-internal ribosomal entry site-Cre
Phosphatidyl inositol-3 kinase
Protein kinase C
Rho guanine nucleotide dissociation inhibitor
Short hairpin RNA sequences against Raptor
Short hairpin RNA sequences against Rictor
Scrambled short hairpin shRNA sequences
Small interfering RNA
Transforming growth factor beta
Triple negative breast cancer
Human epidermal growth factor receptor 2 (HER2) overexpression is a defining feature of approximately 25% of breast cancers and is associated with poor outcome . While HER2-targeted therapies improve outcomes for patients with HER2-amplified breast cancers, resistance to HER2 inhibitors often occurs, underscoring the need for increased understanding of the signaling pathways required for HER2-mediated transformation and malignancy.
The intracellular mTOR kinase exists in two structurally and functionally distinct complexes, defined by the cofactors associated with mTOR, and by their relative sensitivity to rapamycin . Specifically, Raptor is a required cofactor for rapamycin-sensitive mTORC complex 1 (mTORC1), which is activated downstream of phosphatidyl inositol-3 kinase (PI3K)/Akt and mediates cell growth, protein translation, and metabolism . Rictor is a required cofactor for mTORC2, which controls cell survival, polarity, and cytoskeletal dynamics . Control of cytoskeleton reorganization through regulation of the Rho family of GTPases was one of the first described functions of mTORC2  and multiple studies indicate that mTORC2 regulates migration in various cell types including neutrophils, β-cells, endothelial cells, and primary mammary epithelial cells [6, 7, 8, 9].
Given the potent role of motility in tumor progression, it is not surprising that mTORC2 has recently garnered interest for its potential role in cancer metastasis. Studies have shown that luminal breast cancer cells and basal-like breast cancer cells exploit Rictor-dependent mTOR signaling pathways to facilitate invasion and metastasis. For example, small interfering RNA (siRNA)-mediated Rictor knockdown has been shown to inhibit MCF7 (luminal) and MDA-MB-231 (basal-like) breast cancer cell migration [10, 11]. Rictor knockdown inhibits transforming growth factor beta (TGFβ)-mediated epithelial-to-mesenchymal transition (EMT) in basal-like breast cancer lines , and Prickle-dependent cell motility in the MDA-MB-231 model of breast cancer . Additionally, mTOR-independent roles for Rictor in cancer cell migration have also been described. For example, in MDA-MB-231 or T47D (luminal) cells, Rictor was found to interact with protein kinase C (PKC)-ζ to control cancer cell metastasis . However, the contribution of mTORC2 to migration of HER2-amplified breast cancer cells remains unclear. Notably, Rictor mRNA and protein levels are highest in HER2-amplified breast cancers, and correlate with decreased overall patient survival in clinical invasive breast cancer datasets [11, 14]. Rictor depletion from HER2-amplified human breast cancer cell lines or in genetically engineered transgenic mouse models of HER2-amplified breast cancer demonstrates the requirement for Rictor/mTORC2 signaling in tumor formation, tumor cell survival and resistance to HER2-targeted therapy . Given the key role of Rictor in several stages of tumor progression, we were motivated to study the impact of Rictor/mTORC2 in migration, invasion and metastasis of HER2-driven breast cancers.
All animals were housed under pathogen-free conditions, and experiments were performed in accordance with AAALAC guidelines and with Vanderbilt University Institutional Animal Care and Use Committee approval. Rictor FL/FL mice  were kindly provided by Dr. Mark Magnuson (Vanderbilt University) and were inbred to FVB for >10 generations. MMTV-NIC mice (generated in FVB) have been previously described . All analyses of Rictor FL/FL X MMTV-NIC mice were performed on age-matched siblings. For tail vein injections, 5 × 106 SKBR3-scrambled short hairpin shRNA sequences (shScr), SKBR3-short hairpin RNA sequences against Rictor (shRictor), MDA-MB-361-shScr or MDA-MB-361-shRictor cells in 100 μL serum-free DMEM:F12 were delivered to isofluorane-anesthetized mice using a 27-g syringe needle. Freshly harvested Rictor +/+ NIC and Rictor FL/FL NIC tumors were dissociated into single cell suspensions using C-tubes (Miltenyi) for mechanical dissociation, followed by digestion in collagenase A/hyaluronidase (Stem Cell Technologies) for 30 min., 0.05% trypsin-EDTA (Gibco) for 1 min., and DNase I (Stem Cell Technologies) for 10 min. Cells were passed through a 40-μm strainer, and 106 cells were resuspended in 100 μl serum-free DMEM:F12 for delivery to isofluorane-anesthetized wildtype FVB female mice using a 27-g syringe needle.
Lungs were resected from mice and paraffin sections (5 μm) were stained with hematoxylin and eosin (Calbiochem). Immunohistochemical analysis (IHC) on paraffin-embedded sections was performed as described previously  using Rictor (Santa Cruz Biotechnologies) antibodies. Immunodetection was performed using the Vectastain kit (Vector Laboratories), according to the manufacturer’s directions.
In situ Rac-guanosine triphosphate (GTP) assay
Formalin-fixed paraffin-embedded tumor sections were probed 1 h with glutathione-S transferase (GST)-PAK1 Binding Domain (PBD) (Millipore) diluted 1:50 in PBS. GST (lacking PBD) was used as a negative control. Samples were washed then probed with AF488-conjugated anti-GST (1:100), stained with 4',6-diamidino-2-phenylindole (DAPI), and mounted.
BT474, MDA-MB-361, and SKBR3 cells were purchased in 2012 from American Type Culture Collection (ATCC) (cell identity verified by ATCC using genotyping with a Multiplex STR assay) and cultured at low passage in DMEM with 10% fetal calf serum. MCF10A and MCF10A-RictorZFN cells (Sigma-Aldrich) were cultured in DMEM:F12 plus insulin (4 μg/mL), cholera toxin (1 μg/mL), epidermal growth factor (EGF) (100 ng/mL), hydrocortisone (2 μg/mL) and 5% horse serum and transduced with lentiviral HER2-internal ribosomal entry sequence (IRES)-RFP (GenTarget) and selected with 10 ug/mL blasticidin. MMTV-Neu tumor cells were a primary culture of mammary tumor cells derived from a virgin MMTV-Neu female mouse. Tumors were digested in 1X Collagenase A/Hyaluronidase solution (Stem Cell Technologies) for 30 min. at 37 °C, washed five times with serum-free medium, then plated on Matrigel-coated plates in serum-free DMEM:F12 plus insulin (4 μg/mL), EGF (10 ng/mL) and hydrocortisone (2 μg/mL). AZD5363, PP242, and lapatinib were purchased from SelleckChem. The in-solution Rac1 inhibitor was purchased from Calbiochem/Millipore. Adenoviral particles Ad.caRac1, Ad.PKCα, Ad. RFP, and Ad.AktDD were purchased from Vector Biolabs. ARGHGDIB siRNA’s were purchased from Sigma using the following siRNA IDs: SASI_Hs01_00125904 and SASI_Hs01_00125905.
Generation of stable cell lines
Lentiviral shRNA-encoding pLKO plasmids harboring Rictor shRNA (#1853, referred to herein as shRictor.1, and #1854, referred to herein as shRictor.2); scrambled shRNA (#1864) and Raptor shRNA (#1857 referred to here as shRaptor.1 and #1858, shRaptor.2) were transfected into 293FT cells plus packaging vectors. Cultured medium containing viral load was used to infect. Cells were selected and maintained at low passage with puromycin (2 μg/mL). Mouse Rictor was subcloned from pCI-Avo3 (Jacinto et al. ) (Addgene #39210) using high-fidelity PCR and the following primers: forward 5′ CGC AAA TGG GCG GTA GGC TGT and reverse 5′GCT AGT TAT TGC TCA CGC C. Ends were blunted and then cloned into the blunted EcoRI site of pBABE-Puro. Retroviral particles were generated in 239 T cells transfected with pBABE vectors and pCL-Eco. Cultured medium containing viral load was filtered then added to MMTV-Neu cells. At 48 h after infection, MMTV-Neu cells were split 1:5 then selected and maintained with puromycin (2 μg/mL).
Cells were homogenized in ice-cold lysis buffer (50 mM Tris pH 7.4, 100 mM NaF, 120 mM NaCl, 0.5% NP-40, 100 μM Na3VO4, 1X protease inhibitor cocktail (Roche)) and cleared by centrifugation (4 °C, 13,000 × g, 10 min.). Protein concentration was determined using bicinchoninic acid (BCA) assay (Pierce). Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes. Membranes were blocked and probed with antibodies as described previously  using primary antibodies: α-actin (Sigma-Aldrich); Rictor (Santa Cruz); RhoGDI2 (Spring Bioscience); and the following from Cell Signaling Technologies (phospho-cocktail; AKT, P-Akt S473, P-Akt T308, S6, P-S6, and Raptor).
Rac-GTP and Rac-guanine nucleotide exchange factor (Rac-GEF) pull down assays
GST-PBD beads (25 μg; Cytoskeleton #PAK02, resuspended to 1 μg/μL) were mixed with 750 μg cell lysate and rotated end-over-end for 1 h at 4 °C. Beads/lysate mixture was spun at 5000 rpm for 30 sec. Supernatant was removed, beads were washed in PBS, boiled in 13 μL denaturing sample buffer for 10 min., and supernatant was assessed by western analysis using primary antibody against Rac1 (BD Biosciences). The Active Rac-GEF Assay (Tiam1) kit (Cell Biolabs, Inc.) was used according to the manufacturer’s instructions. Briefly, cells at 70% confluence were harvested in the supplied 1X assay buffer, protein content assessed, and 750 μg protein lysate was incubated with 40 μL RacG15A-agarose bead slurry overnight at 4 °C. Beads were washed five times with assay buffer and then assessed by western analysis using the rabbit anti-Tiam1 antibody enclosed in the kit.
Transwell migration/invasion assays
Breast cancer cells were serum-starved overnight, trypsinized, then 100,000 cells were seeded in the upper chamber of Matrigel-coated transwells in serum-free medium, with cells migrating towards the lower chamber in response to 10% serum or EGF (20 nM, R&D Systems). Cells on the lower side of the Matrigel-coated membrane were stained with 0.1% crystal violet after 24 h and enumerated as described previously .
Experimental groups were compared with a control group using Student’s unpaired, two-tailed t test. Multiple groups were compared across a single condition using one-way analysis of variance (ANOVA). Two-way ANOVA was used to compare the response of two agents combined to either of the single agents alone. P < 0.05 was used to define significant differences from the null hypothesis.
Rictor loss decreases metastasis in a genetically engineered mouse model of HER2-driven breast cancer
To confirm that Rictor loss decreases tumor metastasis, we dissociated three independently derived Rictor FL/FL NIC and Rictor +/+ NIC primary tumors into single cell suspensions, then delivered equal numbers of cells to wild-type FVB mice via intravenous injection. Tumor cell suspensions were examined by western analysis to confirm reduction of Rictor protein levels in Rictor FL/FL NIC samples (Fig. 1c), although some Rictor expression remained, perhaps due to incomplete recombination at floxed Rictor alleles, or to an abundance of non-tumor cells in the tumor suspensions that would not be subjected to Cre-mediated recombination. Rictor loss diminished Akt phosphorylation at S473, the mTORC2 phosphorylation site, suggesting that mTORC2 activity was reduced upon Rictor loss in mammary tumors. At 8 weeks after delivery of tumor cells, mouse lungs were assessed for metastatic lesions. Lung metastases were identified in mice receiving Rictor +/+ NIC tumor cells, while Rictor FL/FL NIC tumor cells failed to form metastatic lung lesions (Fig. 1d, N = 3 donor tumors per genotype times 2 recipients per donor tumor).
To determine the impact of increased Rictor expression on mammary tumor metastasis, we transduced primary mammary tumor cells derived from MMTV-Neu mice with pBABE retroviral particles encoding mouse Rictor, followed by puromycin selection. Western analysis confirmed Rictor overexpression in cells expressing pBABE-Rictor, but not in cells expressing empty pBABE (Fig. 1e). Rictor overexpression did not increase P-Akt levels under basal conditions, but prevented P-Akt inhibition following treatment of cells with lapatinib, an HER2 tyrosine kinase inhibitor. We found that intravenous delivery of MMTV-Neu/pBABE and MMTV-Neu/pBABE-Rictor cells generated lung metastases (Fig. 1f). Rictor overexpression in metastases derived from MMTV-Neu/pBABE-Rictor cells was confirmed by IHC. While MMTV-Neu/pBABE cells to wild-type FVB mice resulted in metastatic lung lesions in 41.7% of recipients, consistent with previous reports that MMTV-Neu mammary tumors metastasize to the lungs with a penetrance of approximately 30%, we found that 100% of MMTV-Neu/pBABE-Rictor recipients harbored lung metastases (Fig. 1g, left panel), and on average, harbored nearly twice as many metastatic lesion over recipients of MMTV-Neu/pBABE cells (Fig. 1g, right panel). These data suggest that Rictor promotes metastasis in transgenic mouse models of HER2-overexpressing breast cancer.
Metastasis of HER2-amplified breast cancer cells is reduced upon Rictor loss
The effect of Rictor knockdown in metastasis of HER2-amplified human breast cancer cells was examined by delivering 5 × 106 SKBR3 or MDA-MD-361 cells expressing shScr and ShRictor to athymic Balb/C (nu/nu) mice via tail vein injection. Lungs harvested 8 weeks after tumor cell delivery were assessed in histological sections (Fig. 2g). These studies revealed lung metastases formed by shScr-expressing SKBR3 and MDA-MB-361 cells in 3/7 and 2/10 recipients, respectively (Fig. 2h), suggesting that these cell lines are not robust models of metastasis. Nonetheless, lung metastases were reduced upon Rictor knockdown, being detected in only one of the seven recipients of SKBR3-shRictor cells, and were not detected in any of the 10 recipients of MDA-MB-361-shRictor cells. These findings are consistent with the hypothesis that Rictor regulates metastasis in HER2+ breast cancer cells, and support previous studies demonstrating Rictor-dependent motility in other cancer cell lines, including estrogen receptor (ER)-α- breast cancer MCF7 cells and triple negative breast cancer (TNBC) MDA-MB-231 cells .
Rictor loss decreases Rac1-dependent cell migration and invasion in HER2-amplified breast cancer cells
mTORC2 signaling suppresses RhoGDI2 expression to enhance Rac1-mediated cell motility
Because PP242, like other mTOR kinase inhibitors, inhibits both mTORC1 and mTORC2, it is possible that blockade of mTORC1 accounts partially or in full for impaired dampening of RhoGDI2 expression and decreased Rac1 activation. Therefore, we assessed the impact of selective mTORC1 inhibition on RhoGDI2 expression and Rac1 activity. Although the mTOR inhibitor rapamycin and the related rapalogues exhibit greater selectivity for mTORC1 over mTORC2, accumulating evidence suggests that rapalogues indirectly block mTORC2 activity in many cell types, including mammary epithelial cells. To ensure selective mTORC1 inhibition, we used shRNA sequences directed against Raptor, the mTORC1 obligate cofactor. Previous studies demonstrate that Raptor knockdown blocks mTORC1 signaling. Consistent with this, Raptor knockdown in SKBR3 and MDA-MB-361 cells, which was confirmed by western analysis, resulted in decreased phosphorylation of the mTORC1 substrate p70 S6 Kinase (p70S6K) (Fig. 4e). Phosphorylation of Akt at Ser473, the mTORC2 phosphorylation site, was unaffected by Raptor knockdown, confirming selective inhibition of mTORC1 signaling by Raptor knockdown. Raptor knockdown did not decrease invasion/migration of cells through Matrigel-coated transwell filters, as opposed to what was seen with Rictor knockdown (Fig. 4f-g). Further, RhoGDI2 expression was unaffected by Raptor knockdown in SKBR3 cells (Additional file 1: Figure S3). These results suggest that mTORC2 signaling is distinctly important for supporting migration in HER2-amplified breast cancer cells, due at least in part to regulation of RhoGDI2 levels.
RhoGDI2 suppression increases tumor cell motility and decreases metastasis-free survival
Akt partially rescues migration and invasion of HER2-amplified breast cancer cells lacking Rictor
These data suggest that Rictor/mTORC2 engages Akt signaling to activate Rac1 in SKBR3 and MDA-MB-361 cells, but not through downregulation of RhoGDI2, but rather through activation of Tiam-1. This hypothesis was confirmed using the ATP-competing Akt inhibitor AZD5363, which increases Akt phosphorylation while blocking Akt enzymatic activity. AZD5363 decreased phosphorylation of the Akt substrate glucose synthase kinase (GSK)-3β, decreased active Rac, and decreased active Tiam1 in SKBR3 and MDA-MB-361 cells (Fig. 6c), and substantially reduced cell motility/invasion (Fig. 6d). These results suggest that Rictor-mediated Akt signaling increases Tiam1 activation to support Rac1 activity.
To determine if another mTORC2 effector might be responsible for RhoGDI2 downregulation, we investigated protein kinase C (PKC)α, a direct substrate of mTORC2. Previous studies have shown that PKCα interacts directly with RhoGDI2, causing RhoGDI2 phosphorylation and downregulation. We found that phosphorylation of PKCα was abolished in Rictor-depleted cells (Fig. 6e). We used PKCα overexpression to increase PKCα activity and phosphorylation, resulting in RhoGDI2 downregulation in Rictor-depleted cells, and at the same time, increased Rac-GTP. Further, rescue of PKCα activity increased invasion and motility of Rictor-depleted cells through Matrigel-coated transwell filters (Fig. 6f). These studies suggest that mTORC2 coordinates two signaling pathways, Akt and PKCα, which destabilize the Rac inhibitor RhoGDI2, while using Akt to activate the Rac-GEF Tiam-1, to achieve maximal Rac activation and cell motility, supporting metastasis in HER2-amplified breast cancers (Fig. 6g).
In our previous studies, restoration of Akt signaling was sufficient to fully restore cell survival downstream of Rictor . However, we show here that constitutive Akt signaling only partially rescued the invasion defects caused by Rictor loss. Several lines of evidence suggest that cancer cells exploit Rictor-dependent signaling pathways to facilitate invasion and metastasis. For example, siRNA-mediated Rictor depletion decreases migration of MCF7 and MDA-MB-231 cells [10, 11]. In gliomas, Rictor overexpression promotes mTORC2 activity and tumor cell growth and motility [26, 27]. Herein, we report that HER2-amplified breast cancer cell lines and tumors significantly decrease Rac1 activity upon Rictor knockdown. Recent studies in MEFs showed that Rictor suppresses expression of RhoGDI2, an endogenous Rac1 inhibitor and suppressor of metastasis, thus facilitating Rac1-mediated cell motility . Results shown herein are in agreement with this observation, providing the first validation of this mechanism (Rictor/mTORC2 suppression of RhoGDI2 to activate Rac) in HER2-amplified breast cancer cell migration/metastasis.
Although these data are the first (to our knowledge) linking mTORC2/Rictor to Rac1-mediated invasion and metastasis in spontaneous breast cancer models, Rictor-to-Rac1 signaling has been previously identified in cancers originating from other tissues [28, 29, 30], and previous studies have shown that HER2-amplified breast cancer cells use p120 Catenin-to-Rac1 signaling to promote breast cancer metastasis . These previous reports, taken together with data shown here, highlight the numerous signaling pathways that can converge on Rac1 to promote dissemination of cancer cells . We show evidence that the endogenous Rac inhibitor RhoGDI2 is an important molecular brake for restraining Rac activity. Since RhoGDI2 expression levels correlated significantly with outcome in breast cancer datasets, it is possible that RhoGDI2 downregulation could be used as a predictor of aggressive versus benign disease, although this is a hypothesis that has not yet been tested.
Although Rictor-mediated RhoGDI2 downregulation has been previously reported in MEFs , those studies showed that this is an mTORC2-independent role for Rictor. However, we have shown here that inhibition of mTOR kinase activity using the small molecular weight compound PP242 caused a similar accumulation of RhoGDI2, diminution of Rac-GTP, and blockade of tumor cell motility as was seen upon Rictor knockdown. This suggests that, unlike what occurs in MEFs, HER2-amplified breast cancer cells use Rictor within the context of catalytically active mTORC2 to downregulate RhoGDI2, allowing Rac1 activation and cell motility.
Efficient metastatic progression relies on a coordinated series of steps involving cell motility and invasion, in addition to cell survival and proliferation . While we show that Rictor loss decreases metastatic ability of HER2-driven breast tumors and that Rictor/mTORC2 directly controls cell migration, we have not ruled out the potential impact Rictor loss may have on survival of disseminated tumor cells and how this may affect seeding/establishment of overt metastases in our mouse models of metastasis used for this study. Since previous studies indicate that mTORC2 is required for survival of HER2-amplified breast cancer cells [10, 14], it is likely that the reduced metastases seen upon knockdown of Rictor are due to both a reduction in tumor cell survival and a reduction in tumor cell motility. However, tumor cell survival in Rictor-deficient HER2-amplified breast cancer cells was fully rescued by re-activation of Akt signaling. In contrast, we show herein that Akt re-activation only partially rescued invasion and migration of HER2-amplified breast cancer cells lacking Rictor. Thus, Akt signaling is necessary and sufficient for mTORC2-mediated tumor cell survival, but not for mTORC2-mediated cell motility.
These studies demonstrate the requirement for mTORC2 signaling in HER2-amplified breast cancer metastasis through Rac1. Importantly, these studies demonstrate that Rictor/mTORC2 suppresses the Rac inhibitor RhoGDI2, while activating the Rac-GEF Tiam-1, through two parallel pathways that cooperate to potently activate Rac-dependent cell migration and invasion. These findings support growing literature describing the many pathways that activate mTORC2 and those that are activated by mTORC2. Importantly, evidence of the key roles played by mTORC2 in cancer formation, progression, and metastasis is expanding rapidly.
We would like to acknowledge Dr. Jin Chen, Dr. Jamie Stanford, Dr. David Vaught, and Dr. Carlos Arteaga for their thoughtful discussion of these data. We are grateful for their contributions. We would like to acknowledge the shared resources at Vanderbilt University, Vanderbilt University Medical Center, and the Vanderbilt-Ingram Cancer Center that contributed to the studies reported herein, including the VICC Breast SPORE pathology service under the direction of Dr. Melinda Sanders and the VUMC Translational Pathology shared resource under the direction of Dr. Kelli Boyd.
This work was supported by Specialized Program of Research Excellence (SPORE) grant NIH P50 CA098131 (VICC), Cancer Center Support grant NIH P30 CA68485 (VICC), NIH F31 CA195989-01 (MMW) and CTSA UL1TR000445 from the National Center for Advancing Translational Sciences.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
MMJ performed MMTV-NIC animal studies and performed all transwell migration assays. MJ and BJ generated knockdown of Raptor and Rictor in SKBR3 and MDA-MB-361 cells, and performed their analysis by transwell migration and their analysis by western blot. MMJ and CDY performed PBD pull-down assays. MMW performed some experiments using SKBR3 and MDA-MB-361 cells expressing shRictor. DJH performed animal studies of experimental metastasis. VS performed immunohistochemical analyses of mammary glands. DDS provided assessment capability for RhoGDI2. WJM provided and characterized the MMTV-NIC mouse model. RSC, MMJ, and DB-S designed experiments. RSC and MMJ wrote the manuscript. All authors discussed and interpreted data. All authors read and approved the manuscript prior to submission.
The authors declare that they have no competing interests.
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All animal studies were performed in accordance with protocols reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee, in AAALAC approved animal care facilities.
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