PTEN loss mediated Akt activation promotes prostate tumor growth and metastasis via CXCL12/CXCR4 signaling
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The chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis.
Murine prostate epithelial cells from PTEN+/+, PTEN +/− , and PTEN−/− (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed.
Loss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth.
These results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases.
KeywordsDU145 Cell CXCR4 Expression Human Prostate Cancer Cell PTEN Loss Trabecular Bone Area
Chemokines are a superfamily of cytokines known to regulate the migration of cells and play a key role in the regulation of metastasis. The chemokine CXCL12, also known as stromal-derived factor-1 (SDF-1), is a potent chemoattractant for hematopoetic cells  and activate signaling events through its two distinct receptors, CXCR4 and CXCR7. CXCR4 has been shown to be a key receptor in mediating the metastasis of multiple types of tumors. Binding of CXCL12 to CXCR4 induces trimeric G protein signaling leading to activation of the Src, PI3K/Akt, ERK, and JNK pathways, contributing to protease production and cellular migration and invasion. In addition, we recently found that epidermal growth factor receptor family members are activated downstream of CXCL12/CXCR4 signaling, providing proliferative signals in bone tumor growth. CXCL12 and its receptors have been strongly linked to prostate cancer bone metastasis and are markers for poor prognosis [2, 3, 4, 5].
The tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN, also known as MMAC1/TEP1) is a lipid and protein phosphatase that serves as a negative regulator of the phosphatidylinositol-3 kinase (PI3K) pathway . PTEN dephosphorylates phos-phatidylinositol-3,4,5-trisphosphate (PIP3), thus serving as an inhibitor of the PI3K signaling pathway. Through its attenuation of the PI3K pathway, PTEN is a critical regulator of growth factor signaling and is able to regulate key cellular processes such as cell proliferation, motility, protein synthesis, glucose metabolism, genomic stability, and survival. Mutations of PTEN are associated with several diseases, including Cowden disease, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome [7, 8]. In addition, loss of PTEN has been shown to be associated with many types of cancer, such as glioblastoma, endometrial carcinoma, and breast cancer [9, 10, 11]. PTEN expression is frequently altered in cancer; PTEN is lost or mutated in 50-80% of primary PC, and complete loss of PTEN is associated with aggressive and metastatic cancer [5, 12]. In mouse models of PC, loss of PTEN is critical for tumor initiation, and the level of PTEN expression is inversely associated with prostate tumorigenesis [13, 14]. As shown by microarray analysis and immunohistochemistry, murine epithelial cells from these PTEN-deficient prostate tumors display increased expression of CXCL12 and CXCR4 as compared to the normal prostate glands of PTEN+/+ and PTEN+/− mice . However, it is not known whether Akt activation downstream of PTEN loss regulates CXCL12/CXCR4 expression and function in tumor cells.
In this study, by using cell lines derived from PTEN+/+, PTEN+/−, and PTEN−/− mice, we demonstrate that loss of PTEN results in increased expression of CXCL12 and CXCR4. Using inhibitor assays, we demonstrate that regulation of the PI3K/Akt pathway by PTEN in turn regulates expression of both CXCL12 and CXCR4 in mouse and human prostate cancer cells. Akt overexpression in PTEN wild type DU145 cells induced cell proliferation, tumor growth and bone metastasis. Taken together, these data define a relationship between PTEN loss and CXCL12/CXCR4 signaling in prostate cancer progression.
Materials and methods
Cell lines were cultured in a humidified incubator with 5% CO2 at 37°C. All media were supplemented with 2 mM glutamine, 100units/ml penicillin, and 100 mg/ml streptomycin (Life Technologies Inc., Carlsbad, CA). Murine cell lines were maintained in Advanced DMEM supplemented with 5% fetal bovine serum. The benign human prostate cell line BPH-1 and human PC cell line PC3 were maintained in RPMI-1640 supplemented with 10% fetal bovine serum. Human PC cell line C4-2B and DU145 were maintained in T-Medium supplemented with 10% fetal bovine serum and DMEM supplemented with 10% fetal bovine serum, respectively.
Establishment of PTEN+/+, PTEN+/−, and PTEN−/− mouse prostate epithelial cell lines, DU145-neo and DU145-HA-Akt1 stable cell lines
Murine cell lines were established as described previously [16, 17]. Briefly, exon 5 of PTEN was deleted specifically in the murine prostate. PTEN+/+, PTEN+/−, and PTEN−/− prostate epithelial cells were isolated from prostates of corresponding mice at 8 weeks of age, and cell lines were established by serial dilution method and subsequent clonal selection. DU145 cells were transfected with PLNCX-neo and PLNCX- Hemagglutinin-tagged Akt1 constructs using lipofectamine 2000; 48 hours post-transfection, cells were exposed to Neomycin, and stable clones were selected.
Western blot analysis
Cells were washed with PBS, and total cellular proteins were extracted with buffer containing 62.5 mM Tris–HCl (pH 6.8), 2% SDS, 1 mM PMSF, and 1X Protease inhibitor cocktail (Roche, Indianapolis, IN). Protein content was quantified with a BCA protein assay (Pierce Biotechnology, Inc, Rockford, IL), and equal amounts of protein were resolved by 10% SDS-PAGE. Immunoblot was performed with antibodies against PTEN, phosphorylated Akt (S473), and total Akt (Cell Signaling Technology, Boston, MA), CXCR4 (Chemicon, Billerica, MA) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). The band intensities were determined by quantitation of pixel intensities using ImageJ software (version 10.2; National Institutes of Health, Bethesda, MD).
mRNA was purified from cells using the RNeasy kit (Qiagen, Valencia, CA), and cDNA synthesis was performed with iScript Select cDNA Synthesis Kit (Biorad, Hercules, CA). Real time RT-PCR was performed using SYBR Green mix plus ROX (Fisher Scientific) and the Eppendorf Mastercycler ep realplex2 qPCR System (Hauppauge, NY) according to the manufacturer’s protocol. Relative values of gene expression were normalized to GAPDH and calculated using the 2-ΔΔCt method, where ΔΔCt = (ΔCttarget gene - ΔCtGAPDH)sample - (ΔCttarget gene - ΔCtGAPDH)control. The fold change in relative expression was then determined by calculating 2-ΔΔCt. Forward and reverse murine specific primers used are as follows: CXCR4: 5′-TCAGTGGCTGACCTCCTCTT-3′, 5′-TTTCAGCCAGCAGTTTCCTT-3′; CXCL12: 5′-CTTCATCCCCATTCTCCTCA-3′, 5′-GACTCTGCTCTGGTGGAAGG-3′. Forward and reverse human specific primers used are as follows: CXCR4: 5′-GGTGGTCTATGTTGGCGTCT-3′, 5′-TGGAGTGTGACAGCTTGGAG-3′; CXCL12: 5′-ATGAACGCCAAGGTCGTG-3′, 5′-CTTCGGGTCAATGCACACTT-3′. Forward and reverse primers recognizing both murine and human GAPDH were 5′- ATCACCATCTTCCAGGAGCGA-3′ and 5′-GCCAGTGAGCTTCCCGTTCA-3′, respectively.
Inhibition of Akt signaling pathway
Cells were cultured with growth media supplemented with 1% FBS and treated with indicated concentrations of Akt Inhibitor IV (Fisher Scientific, Pittsburgh, PA) or vehicle control for 18 hours. Subsequently, mRNA and protein were collected from cells and subjected to quantitative RT-PCR analyses or western blot analysis, respectively.
Cells were cultured with complete growth media and treated with 10 μM Akt Inhibitor IV or vehicle control; after five hours, media was replaced with growth media supplemented with 1% FBS containing 10 μM Akt Inhibitor IV or vehicle control. After overnight culture, cells were trypsinized and plated on the upper chamber of matrigel-coated transwell filters (2x105 cells/filter) in growth media supplemented with 1% FBS containing 10 μM Akt Inhibitor IV or vehicle control, with 200 ng/mL CXCL12 added to bottom chamber. After 24 hours, cotton swabs were used to remove unmigrated cells from the upper chamber, and inserts were stained with 0.9% crystal violet. Total number of migrated cells was counted under 10× magnification. Assay was performed in triplicate.
In vivo studies and tumor tissue analyses
Both subcutaneous and intratibial tumor inoculation studies were performed as described previously . Briefly, five-week-old male C.B.-17 severe combined immunodeficient (SCID) mice (Taconic Farms, Germantown, NY) were used in the study. For subcutaneous tumor cell implantation, 5x105 cells of both DU145-Neo and DU145- Hemagglutinin-tagged AKT1 transfectants were mixed in 50% matrigel in a volume of 100 μl and implanted in flanks of mice. For each cell line, eight mice were used in the experiment. For intratibial implantation 1x105 cells were injected per bone; for each cell line, 8–10 mice were used. Histomorphometric analyses were performed to determine tumor burden and trabecular bone area in both DU145 transfectants (Neo and HA-Akt1) as previously described .
Formalin-fixed, paraffin-embedded serial tissue sections from DU145-Neo and DU145-HA-Akt1 tumors were deparaffinized with xylene and rehydrated in graded EtOH. Endogenous peroxidase activity was blocked by incubating in 3% H2O2 for 20 min. For subcutaneous tumor sections, antigen retrieval was performed with An-tigen Retrieval Citra Plus Solution (BioGenex, Freemont, CA) in a steamer. For bone sections, antigen retrieval was performed with proteinase K (Sigma-Aldrich, St. Louis, MO). Slides were then blocked with Blocking Serum from ABC Vectastain Kit (Vector Labs, Burlin-game, CA). Slides were incubated at 4°C overnight in a humidified chamber with antibodies directed against Ki67 (BD Biosciences, San Jose, CA), phosphorylated Akt (S473) (Cell Signaling Technology), or CXCR4 (R&D Systems, Minneapolis, MN). After washing, sections were incubated with ABC Vectastain Kit, according to manufacturer’s protocol, followed by incubation with 3,3-diaminobenzidine tetrahydrochloride (Vector Labs). Nuclei were counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Sections were then dehydrated with graded EtOH, washed with xylene, and mounted with Permount (Sigma-Aldrich).
Data were analyzed using Microsoft Excel 2008. All data are presented as mean ± SD. Data were analyzed using Student’s t-test; a p-value < 0.05 was considered statistically significant.
Progressive loss of PTEN results in increased expression of CXCL12 and CXCR4 in murine prostate epithelial cells
Akt regulates CXCR4 expression in PTEN-null human prostate cancer cells
Overexpression of Akt results in increased phosphorylation of Akt, CXCR4 expression, proliferation and invasion
Overexpression of Akt results in increased subcutaneous tumor growth
Overexpression of Akt1 results in increased intratibial tumor growth
Chemokines and their receptors play key roles in hematopoietic cell trafficking. CXCL12/CXCR4 has also been shown to play a key role in the regulation of metastasis, and its expression has been shown to be elevated in localized and metastatic cancer, including bone metastatic prostate tumors [2, 3]. Among PC patients, higher expression of CXCR4 was documented in prostate tumor tissues from African American patients, suggesting CXCR4 expression is associated with aggressive disease phenotypes in these patients . Human prostate tumor expression of CXCR4 is also associate with poor survival , as its expression is significantly associated with local recurrence after therapy and formation of distant metastases . Taken together, these studies emphasize the clinical significance of CXCL12/CXCR4 expression in prostate tumor progression.
Tumor cells expressing CXCR4 metastasize to target organs that express high levels of CXCL12 [26, 27]. Colonization of tumor cells in the bone microenvironment is thought to occur through PC cell encroachment of the hematopoietic stem cell niche by mimicking the stem cell interactions with bone resident stromal cells . This concept is validated by studies showing that targeting CXCR4 function through neutralizing antibodies inhibited prostate cancer bone metastasis , and overexpression of CXCR4 in prostate cancer cells enhanced bone metastasis . Additionally, inhibition of CXCR4 with CTCE-9908 resulted in decreased tumor growth, angiogenesis, and lymphangiogenesis, as well as increased apoptosis in a xenograft PC model . Thus, these studies show that the CXCL12/CXCR4 axis in tumor cells usurps stem cell homing mechanisms to get into bone [28, 29] and subsequent colonization and growth through activation of growth factor receptor signaling . Studies with colorectal cancers show that CXCR4 signaling is also involved in outgrowth of metastasis . Taken together, these studies demonstrate that CXCL12/CXCR4 signaling is a critical event mediating homing of tumor cells and subsequent expansion of metastases.
Our previous studies show that PTEN knockout mouse model epithelial tumor cells gain expression of an “osteogenic signature,” thus predisposing these cells for metastasis . We found that both CXCL12 and CXCR4 are overexpressed in epithelial tumor cells in PTEN knockout mice. Herein, using cultured cells from PTEN intact, heterozygous, and knockout cells, we show that both CXCR4 and CXCL12 expression is higher in PTEN knockout cells, thus confirming the immunohistochemical tumor expression findings (Figure 1). Specific inhibition of Akt resulted in downregulation of both CXCL12/CXCR4 expression, and an established PI3 kinase inhibitor also downregulated both CXCL12 and CXCR4 gene expression (data not shown). Similarly, Akt-mediated regulation of CXCR4 was observed in human prostate cancer cells with loss or mutation of PTEN (Figure 2). Additionally, in both mouse and human tumor cells with either loss or mutation of PTEN, Akt Inhibitor IV treatment inhibited basal as well as CXCL12-induced invasion. PTEN loss-induced PI3K/Akt has been shown to mediate migration and invasion of prostate cancer cells in response to CXCL12/CXCR4 [20, 33, 34], suggesting that Akt can function both upstream (as an inducer of CXCR4 expression) and downstream (as a signaling kinase for induction of proteases and invasion) of CXCR4. Current studies with Akt inhibitors implicate Akt as a key member in this pathway contributing to CXCR4 expression in prostate cancer cells.
Tumor suppressive functions of PTEN independent of phosphoinositide lipid phosphatase activity play a key role in cell cycle regulation, maintaining genomic instability, and controlling DNA repair mechanisms that safeguard cells from accumulation of genetic mutations and uncontrolled proliferation . PTEN loss dysregulates these genome safeguard mechanisms and also leads to Akt activation, promoting tumorigeneisis. To determine the function of Akt in tumor growth and metastasis via regulation of CXCR4 function independent of other effects induced by PTEN loss, DU145 cells were transfected with an HA-tagged Akt1, which is not constitutively active and must be activated within the cell (Figure 3). Akt1 overexpression resulted in increased proliferation and cell cycle progression, suggesting that transfected Akt1 mimicked PTEN loss-associated hyperactivated Akt signaling. In an effort to determine the biological significance of Akt/CXCR4 axis in tumor growth, we subcutaneously implanted both low Akt1 (Neo) and high Akt1 (HA-Akt1) expressing cells in SCID mice (Figure 4). Akt1 overexpression significantly enhanced tumor growth starting from day 60. Immunohistochemical analysis further revealed that HA-Akt1 tumors proliferated at a faster rate, as shown by increased staining for Ki-67 proliferation marker. As expected, HA-Akt1 tumors have stronger Akt Serine 473 phosphorylation and CXCR4 expression, suggesting that Akt1 induced CXCR4 expression, contributing to the primary tumor growth. Our previous data demonstrate that CXCR4 overexpression in the PTEN-null cell line PC-3 enhanced bone tumor growth in a SCID-human model ; these tumors have activated Akt signaling, demonstrating the Akt signaling downstream of CXCR4 contributing to bone tumor growth. Herein, we utilized DU145-HA-Akt1 overexpressing cells to specifically determine the contribution of Akt1 without perturbing the lipid/protein phosphatase functions of PTEN in CXCR4 expression and downstream signaling in bone tumor growth. Activation of Akt1 in these cells was enhanced by bone factors and/or bone stromal cell interactions. In intratibial models, DU145 cells produce osteosclerotic reactions, as evidenced by enhanced bone formation measured by X-rays and histomorphometry. DU145-Neo cells produced similar bone reactions in our model, whereas overexpression of Akt1 completely reversed this phenotype to an osteolytic phenotype similar to PC-3 cells in this model, as evidenced by enhanced tumor growth and destruction of trabecular bone (Figure 5). Akt1 activation, as measured by Serine 473 phosphorylation, is higher in DU145-HA-Akt1 cells and is localized to the bone tumor interface, suggesting that transfected Akt1 is activated by bone remodeling-released and/or stromal-expressed factors. CXCR4 expression is also enhanced in bone tumors (Figure 5E), suggesting that CXCL12/CXCR4 signaling contributing to bone tumor growth in Akt1 transfected DU145 cells.
In summary, these data showed that PTEN loss-induced PI3K/Akt pathway induces CXCL12/CXCR4 expression, and this expression is particularly mediated by Akt kinase in both murine and human prostate cancer cells. Akt1-induced CXCR4 expression is active in CXCL12-induced cellular invasion, tumor growth, and intraosseous tumor growth in murine model systems. Given the frequency of PTEN gene alterations in advanced human prostate tumors and expression of CXCR4 in these patients, Akt1 signaling may be a therapeutic target for advanced prostate cancer patients.
We would like to thank Louie Semaan and Li Yanfeng for excellent technical assistance and Dr. R. Daniel Bonfil for providing technical assistance on bone tumor immunohistochemical analyses. Sreenivasa R. Chinni is supported by U.S. Department of Defense, Idea Award W81XWH-09-1-0250 and NIH R01CA151557 for collection, analysis and interpretation of data.
Supported by U.S. Department of Defense, W81XWH-09-1-0250, NIH-NCI Grant CA151557 and NIH P30 CA22453.
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