Background
Breast cancer is the second most common cause of cancer death in women across the world. Most of these deaths occur due to metastatic spread and disease relapse. Breast cancer is a heterogeneous disease and transcriptome profiling has led to the identification of at least six molecular subtypes of the disease, including luminal A, luminal B, Her2-enriched, basal-like, claudin-low, and normal-like [
1]. Importantly, each of these subtypes is associated with varying prognoses and has differential sensitivities to conventional therapies. For example, the basal and claudin-low subtypes are known to have poorest outcomes in patients as compared to the other subtypes but do not respond to hormonal or HER2-targeted therapy [
2]. This highlights the necessity of finding novel treatment strategies to target basal-like breast cancers, since effective treatment options are still lacking.
Wnt proteins are a family of ligands that bind to Frizzled receptors and initiate an intracellular signaling pathway leading to activation of various genes related to developmental pathways. The canonical Wnt pathway involves the stabilization of β-catenin, which can enter the nucleus and transactivate Wnt target genes [
3]. This canonical Wnt pathway has been implicated in human basal-like breast tumors. Wnt-1 (initially named as int-1), a member of the family of Wnt ligands, was the first protooncogene identified to be activated by the nearby insertion of mouse mammary tumor virus (MMTV) proviruses in mammary tumors of infected mice [
4]. Transgenic expression of Wnt1 using MMTV-LTR enhancer is sufficient to form mammary adenocarcinomas. The MMTV-Wnt1 mouse model develops mammary tumors which can be predominantly classified as the basal subtype. These tumors are enriched for mammary stem cells (MaSC) and predominantly express basal cell markers [
5]. Transcriptomic profile of these tumors resembles a MaSC-like signature [
6]. Hence, MMTV-Wnt1 serves as a basal-like breast cancer mice model to study the relevance of various signaling pathways active in human basal-like breast cancer.
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is activated downstream of integrin receptors in response to binding to the extracellular matrix [
7]. Other than ECM receptors, FAK also plays a role as a mediator of cell signaling downstream of growth factor and cytokine receptors. FAK is highly expressed in a number of cancers including breast, intestinal, and ovarian, in which it is known to promote cancer growth and metastasis through both kinase-dependent and independent mechanisms [
8]. Previous studies have shown that FAK plays an important role in promoting mammary tumor development and progression in mouse models of luminal B [
9‐
12] and HER-2 [
13] subtypes of breast cancer. Additionally, FAK is highly expressed in triple-negative and basal-like breast cancer [
14,
15]. A recent study showed that FAK also contributed to the malignancy of a human triple-negative breast cancer cell line MDA-MB-231 [
16]. However, direct evidence for the in vivo importance of FAK in basal-like breast cancer is still lacking. Hence, we evaluated the effect of deletion of FAK and disruption of its kinase function in a basal-like breast cancer model.
FAK has been shown to control tumor cell survival, proliferation, and migration through both kinase-dependent and independent pathways. FAK can directly phosphorylate Src family kinases and form a heterodimer complex with Src [
17], which leads to the phosphorylation of p130Cas adaptor molecule and activation of multiple downstream signaling events, including the RAS-Erk pathway in PyMT induced mammary tumors [
18]. Reduction in cyclin D1 levels in PyMT tumors has been associated with reduction in tumor cell proliferation upon loss of FAK [
9]. Kinase function of FAK has also been shown to activate the PI3K-Akt pathway, which can protect cells from apoptosis and promote survival [
8]. FAK can also promote survival through its kinase-independent scaffolding function in the nucleus by promoting MDM2-mediated degradation of p53 [
19].
One of the direct consequences of PI3K-AKT pathway is downstream activation of mTOR signaling. mTOR is a large protein kinase associated with different protein partners to form two independently regulated hetero-oligomeric complexes, the rapamycin-sensitive and rapamycin-insensitive mTOR complex (mTORC) 1 and 2, respectively [
20]. AKT inhibits TSC2 by phosphorylating it, leading to activation of Rheb-GTPase which then directly binds and activates mTORC1. mTORC2 on the other hand can be directly activated by AKT [
21]. mTORC1 can activate protein translation and rewire cellular metabolism, thereby promoting cell growth and survival. It is highly activated in a wide variety of cancers [
22]. Thus, FAK could also affect the mTOR pathway through its activation of PI3K-AKT signaling. Indeed, a recent study showed that FAK inhibition reduced mTOR activation in MCF7 and MDA-MB-231 cells [
23]. Our lab has also previously found that FAK directly interacts with TSC2 and promotes S6 kinase phosphorylation [
24]. Because FAK can activate a number of downstream effectors, it is important to study the contributions of these various pathways in mediating FAK regulation of basal-like breast cancer.
In this study, we demonstrate that though FAK is dispensable for the onset of Wnt1-driven mammary tumors, disruption of FAK’s kinase activity suppressed Wnt1-driven tumor growth and progression through compromised tumor cell survival. We found that FAK activates the AKT-mTOR pathway in these tumors, which in turn supports the survival of the tumor cells. In summary, our studies show that in a basal-like mammary tumor model, FAK is required for survival of the tumor cells and could potentially serve as a therapeutic target in the treatment of basal-like breast cancer.
Methods
Mice
FAK Ctrl (FAK f/f), cKO (FAK f/f, MMTV-Cre), and cKD (FAK f/KD, MMTV-Cre) transgenic mice have been described previously [
9,
25‐
27]. MMTV-CRE Line F mice were obtained from NCI [
28]. MMTV-Wnt1 mice were obtained from Dr. Yi Li [
29] and were crossed with FAK Ctrl, cKD, and cKO mice. We previously showed that the MMTV-Cre targets both the luminal and the basal compartment of the mammary glands [
9,
27,
30]. Our group generated the kinase defective knock-in allele of FAK (KD) by mutating K454 to R in the catalytic domain of FAK (in exon 16 of FAK genomic DNA), and these were initially described in [
26]. Mice were palpated every 7 days after weaning, and the size of tumors was measured with a caliper and recorded. Mice were housed and handled according to local, state, and federal regulations, and all experimental procedures were carried out according to the guidelines of the Institutional Animal Care and Use Committee at the University of Cincinnati.
Reagents and antibodies
Antibodies used in this study include FAK (CST-3285), phospho-FAK Y397 (CST 8556), GAPDH (CST 2118), Ki67 (Spring Bioscience m3062), cleaved caspase 3 (CST 9661), CD31 (Dianovo, DIA310), CD8 (Invitrogen MA1-80231), Vinculin (Sigma V4505), phospho-4EBP1Ser65 (CST 9451), phospho-4EBP1Thr37/46 (CST 2855), 4EBP1 (CST 9644), p-S6K (CST 9205), S6K (Santa Cruz SC-230), phospho-AKT Ser473(CST 4060), phospho-AKT Thr308 (CST 5473), and AKT (CST9272). Inhibitors used in this study include PF-562271 (Cayman), PP242 (MedChem Express), thapsigargin (Cayman), and tunicamycin (Cayman).
Cell culture, treatments, transfection, and transduction of cells
Tumor cells derived from FAK f/f Wnt1 tumors were cultured in DMEM/F12 supplemented with 10% FBS, 10 ng/ml EGF, 20 μg/ml insulin, and 50 units/ml penicillin-streptomycin. Lentivirus production and transduction of the tumor cells with Cre-ERT were carried out as described previously [
31]. Transfection experiments were carried out using Lipofectamine 2000 Reagent (Invitrogen). Deletion of FAK was induced by culturing with 100 nM 4-hydroxy-tamoxifen. Amino acid starvation of the tumor cells was carried out by culturing them for 48 h in HBSS. MDA_MB231, CAL1851, and HCC186 were cultured in DMEM medium supplemented with 10% FBS and 50 units/ml penicillin-streptomycin. Viability of cells after treatment was determined using Alamar Blue reagent (Thermo Fisher).
Migration, wound healing, and sphere formation assay
Cells were seeded at a density of 25,000 cells/well in Boyden chambers coated with growth factor reduced Matrigel (BD Biosciences) and incubated for 24 h. Cells on the membrane were then fixed with ice cold ethanol and stained with crystal violet. Cells which have invaded to the lower side of the membrane were then quantified. For wound healing assay, 10,000 tumor cells were plated per well in a 96-well plate. Wounds were made with Woundmaker (Essen Biosciences) and imaged using Incucyte (Essen Biosciences). Quantifications were done using ImageJ software. For sphere formation assay, tumor cells were plated at density of 10,000 cells/ml in a 96-well low attachment plate and cultured in MEBM medium supplemented with 0.2% B-27, 20 ng/ml EGF, 5 μg/ml insulin, 20 μg/ml Gentamycin, and 0.5 μg/ml Hydrocortisone for 7–10 days.
Flow cytometry
Unattached dead cells and attached cells after treatment were collected after brief trypsinization and stained using BD Pharmingen AnnexinV apoptosis detection kit as per manufacturer’s protocol. Stained cells were analyzed using FACSAria. Flow cytometry data were analyzed using FlowJo software.
METABRIC dataset [
32] was downloaded from cBioportal [
33]. PTK2 gene was queried on cBioportal, and K-M survival plot was visualized and downloaded. Gene amplification and mRNA expression data was analyzed using GraphPad Prism and MS Excel.
RNA sequencing of tumor cells
RNA sequencing experiments were performed by the Genomics, Epigenomics and Sequencing Core in University of Cincinnati. Briefly, RNA from tumor cells was isolated using mirVana miRNA Isolation Kit (Thermo Scientific) according to the manufacturer’s instructions for total RNA isolation. Targeted RNA enrichment was achieved using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs) and PrepX mRNA Library kit (WaferGen) combined with Apollo 324 NGS automated library prep system was used for library preparation. Cluster generation and HiSeq sequencing were carried out using the cBot and HiSeq systems (Illumina) respectively. To analyze differential gene expression, sequence reads were aligned to the genome using standard Illumina sequence analysis pipeline, which was analyzed by The Laboratory for Statistical Genomics and Systems Biology in the University of Cincinnati. The RNA-seq data have been deposited in the GEO database under accession code GSE146659.
Mammary gland whole mounts, histology, and immunohistochemistry
Fourth abdominal mammary glands were excised at 5 weeks after birth, and whole mounts stained with carmine alum were analyzed, as described previously [
27]. Mammary tumors or lungs were harvested from mice and subjected to analysis by histology, immunohistochemistry, as described previously [
9,
34]. Briefly, tumors were fixed overnight in 10% phosphate-buffered 10% formalin (Fisher Scientific), dehydrated in alcohol gradients, xylene, and paraffin before being embedded. Next, they were sectioned into 5-μm-thick slices. Unstained tissue sections were first deparaffinized in xylene (3 times, 5 min each), rehydrated in graded ethanol solutions (100, 95, 70%, 50%, and 30%), and stained with H&E. For immunohistochemistry, tumor sections were first subjected to antigen retrieval (sodium citrate buffer, pH − 6.8), stained with different antibodies and mounted with Permount mounting medium (Fisher chemicals). For quantification of metastatic nodules, we obtained three sections from each lung tissue that were 200 μM apart. All three sections were stained with H&E and scanned through a light microscope at × 4 magnification. A number of metastatic nodules were identified based on histology and counted.
Immunoblotting
Lysates were prepared using modified RIPA buffer as described previously [
27] with the addition of Halt protease and phosphatase inhibitors (Thermo Scientific). Protein concentrations were then quantified by the BCA method, subjected to SDS-PAGE, and analyzed by immunoblotting as described previously [
27].
Statistics
Data were plotted as means ± SEM, and statistical significance was determined using a two-tailed t test, one-way ANOVA, or two-way ANOVA followed by Tukey’s multiple comparison test wherever applicable. For tumor-free survival curves, statistical significance was determined using a log-rank test (Mantel-Cox). The threshold for significance of p values was 0.05.
Discussion
Basal-like breast cancer is an aggressive subtype of breast cancer with limited treatment options [
2]. It has been reported that FAK protein level and its phosphorylation are highly elevated in triple-negative breast cancers [
14,
15], which include basal-like subtype. Consistent with these studies, we found a greater proportion of basal-like tumors as compared to any other subtypes in the METBRIC dataset, to contain genetic FAK amplification and/or high levels of FAK mRNA. To study the mechanistic relevance of these associations in vivo, we knocked out FAK and disrupted its kinase function through a knock-in mutation, in the Wnt1-induced basal-like breast cancer mice model. We found that indeed FAK plays a prominent role in the growth of these tumors. Previously, we and others showed that FAK deletion in MMTV-PyMT tumors, classified as luminal B subtype, suppressed tumor growth [
9], and our studies also showed defective maintenance of mammary cancer stem cells in the mutant mice [
27]. Other studies showed that FAK plays an important role in ERBB2 (HER2+ subtype)-induced mammary tumorigenesis [
13]. Our results provide direct evidence of the importance of FAK in a basal-like breast cancer model and complement previous studies to support FAK as a potential therapeutic target for all major subtypes of human breast cancer.
While FAK promotes mammary tumor growth and metastasis in all models of different subtypes, there were several notable differences between our findings in mouse model for basal-like breast cancer subtype and previous studies in models of other subtypes. As in other models and consistent with a role for FAK in regulating cell migration in many cell types [
9,
25], we found reduced cell migration upon FAK deletion in Wnt1-driven mammary tumor cells. In addition, we also found reduced tumor sphere formation in Wnt1-driven mammary tumor cells upon loss of FAK, as in PyMT-driven tumor cells we reported earlier [
9]. However, FAK ablation decreased the proliferation of mammary tumor cells derived from some other mouse models of breast cancer [
13,
27,
38]. FAK deletion in Wnt1-driven mammary tumor did not change tumor cell proliferation but increased apoptosis which likely also contributes to the reduced tumor growth and metastasis in this model. The other surprising finding is a lack of effect on mammary tumor development upon FAK deletion in this model, despite the apparently significant inhibition of tumor growth and metastasis after tumor appearance. It is possible that the increased apoptosis following FAK deletion or the loss of its kinase activity only occurs when mammary tumors reach palpable size (i.e., tumor appearance as defined in our assays, see Fig.
2) in cKO-Wnt1 and cKD-Wnt1 mice. Interestingly, we only detected reduced survival of iKO-Wnt cells vs iCtrl-Wnt cells under ER stress conditions, but not normal culture conditions, in vitro. Such stress conditions (ER stress or potentially other stresses) may only exist when the tumor reach palpable size in vivo, which could explain an effect of FAK deletion in tumor growth and metastasis at later stage due to increased apoptosis. Interestingly, we found a modest difference in tumor initiation times between the cKO and cKD tumors. Previously, we have reported that the kinase function of FAK regulates luminal progenitors [
27]. However, mammary stem cells (MaSCs) were regulated by a kinase independent function and not dependent on the kinase function of FAK. In the MMTV-Wnt1 model, expansion of MaSCs has been well documented. Thus, the loss of the kinase-independent function of FAK in cKO-Wnt1 mice, but not cKD-Wnt1 or Ctrl-Wnt1, which could compromise the ability of MMTV-Wnt1 induced expansion of MaSCs, may explain the trend for the delayed tumor initiation times in cKO-Wnt1 tumors relative to cKD-Wnt1 and Ctrl-Wnt1 tumors.
Interestingly, there is a large change in cell viability upon treatment with FAK inhibitor (Fig.
6b), as compared to when FAK is ablated in vivo (see Fig.
3d). This could be due to differences between acute inhibition of FAK and constitutive deletion of FAK. For the tumors in vivo, the cells have coped with FAK ablation for long periods of time (since pre-neoplastic stages) and thus may exhibit less dependence on FAK. The doses of FAK inhibitor used in these studies were appropriate for in vitro treatments and had been used as per previous studies [
39,
40].
Gunther et al. showed that Wnt1-initiated mammary tumors require Wnt signaling for tumor maintenance and the tumors regress if Wnt signaling is blocked [
41]. Previously, FAK has been reported to promote Wnt signaling in colorectal cancer by phosphorylating GSK3β thereby blocking the degradation of β-catenin and its accumulation [
42]. However, we did not find any changes in β-catenin levels upon blocking FAK signaling (data not shown). This illustrates the possibility of requirement of independent signaling pathways activated by FAK other than the Wnt pathway in maintaining tumor growth. Based on an unbiased transcriptomic analysis of isogenic Wnt1-driven mammary tumor cells with or without FAK, we found that mTORC1 signaling, ribosome biogenesis, G2-M checkpoint, and E2F target-related genes were significantly downregulated in the absence of FAK. E2F has been previously reported to be an upstream activator of mTOR [
43]. In addition, mTOR has been indicated to control multiple steps in ribosome biogenesis [
44]. These studies further indicate that mTOR and its related pathways might be downregulated as a result of FAK deletion. We further showed that loss of FAK disrupts phosphorylation of AKT at Serine 473 and Threonine 308. Phosphorylation of AKT at Serine 473 and Threonine 308 is usually mediated by mTORC2 and PDK1 respectively. Previously, we have shown that FAK binds PI3K and regulates its activity [
45]. Hence, our results indicate that AKT is regulated downstream of FAK through both the PI3K-PDK1 and mTORC2 cascade. In addition, we found decreased mTORC1 signaling, which is consistent with the reduction in AKT activity and the data from our transcriptomic analysis. The mTOR signaling pathway plays a critical role in mediating growth stimulatory pathways in Wnt tumors [
46]. Indeed, we found that mTOR inhibitor PP242 targeting both mTORC1 and mTORC2 could induce apoptosis in Wnt1-driven mammary tumor cells, supporting a role for mTOR to at least partially mediate FAK regulation of tumor growth and metastasis.
Our studies also showed that in the absence of FAK, the tumor cells were sensitive to ER stress-inducing agents. A recent study suggested that FAK protects endothelial cells from ER stress-induced mitochondrial damage and cell death by activating STAT3 [
47]. This raises the possibility that the absence of FAK confers a survival vulnerability of the tumor cells to induction of ER stress. Whether FAK can directly trigger phosphorylation of STAT3 or if there are other mediators involved in this process needs to be investigated in order to gain more mechanistic insights about the role of FAK in mediating cell survival under ER stress. Thus, we have unraveled a potential combinatorial therapeutic strategy of FAK ablation along with induction of ER stress in Wnt1-driven tumor cells.
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