Background
Prostate cancer (PC) is the most commonly diagnosed solid malignancy in men, with over 192,000 new cases yearly, and the second leading cause of cancer-related death in men in the US, with over 27,000 deaths each year. PC has a specific propensity to metastasize to bone; in fact, in the majority of cases, bone metastases develop long before metastatic growth is apparent in soft viscera. Bone metastases cause pain, compression fractures, spinal cord compromise and other complications. Bone metastasized cancer cells induce bone turnover by recruiting bone resident osteoclasts and osteoblasts, and the resultant bone turnover enhances tumor growth by creating a vicious cycle [
1]. Cancer cells use similar mechanisms as hematopoietic stem cells in homing to bone by competing for the occupation at osteoblastic niches in bone tissue [
2], where chemokine signaling plays a key role in attracting cells to the bone microenvironment [
2‐
4].
The CXCL12/CXCR4 axis has been involved in homing of breast [
5] and prostate [
4,
6] cancer cells to bone where cancer cells have aberrant expression of CXCR4, the receptor for the CXCL12 chemokine [
7‐
9]. Transcriptional regulation of the CXCR4 gene is a key determinant of net cell surface expression of CXCR4 and its subsequent function in transformed epithelial cells and cancer cells. We showed that TMPRSS2-ERG fusions regulate CXCR4 expression in prostate tumors; thus, androgen induced ERG expression transcriptionally regulates CXCR4 expression in PC cells [
7,
9]. In addition, several factors and organ microenvironments have been shown to regulate CXCR4 expression in tumor cells [
10‐
17]. The mammary fat pad and bone microenvironment have been shown to induce CXCR4 gene expression in cancer cells [
16,
18]. At the cellular level, osteoblasts, stromal cells and endothelial cells all express CXCL12 [
4,
6,
10,
19] and contribute to bone metastasis of PC cells [
4,
6].
CXCR4 expression increases during progression of PC; localized prostate carcinoma and bone metastasis tissue express significantly higher levels than benign prostate tissue [
20,
21]. Among PC patients, higher expression of CXCR4 was documented in prostate tumor tissues from African Americans [
22], suggesting aggressive phenotypes often associated with higher CXCR4 expression. CXCR4 expression is associated with shorter progression free survival in cancers [
23], and in prostate cancers its expression is significantly associated with metastatic disease and poor survival [
24,
25]. The chemokine CXCL12 is also over-expressed in PC metastatic tissue compared to normal tissue [
20]. The CXCL12/CXCR4 axis has been shown to play an important role in PC cell proliferation, migration and invasion [
4,
6,
20,
26‐
30].
Previously, we showed that activation of the CXCL12/CXCR4 axis transactivates HER2 [
3,
6] and promotes intraosseous tumor growth [
3]. To further explore the transactivation of HER2 by CXCL12, we investigated the role of the small GTP protein G
αi2 in Src and HER2 phosphorylation in lipid raft membrane microdomains and the significance of CXCR4 inhibition by plerixafor, a bone stem cell mobilizer, in prostate cancer bone tumor growth. Given the important role of CXCL12/CXCR4 signaling in PC bone metastases, our data suggest that CXCL12/CXCR4 inhibition may impact the development of bone metastasis.
Methods
Cell culture
Cell lines were cultured in a humidified incubator with 5 % CO2 at 37 °C. All media were supplemented with 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies Inc., Carlsbad, CA). PC3 cells maintained in RPMI-1640 supplemented with 10 % fetal bovine serum (FBS), PC-3 M-luc2 cells in EMEM medium supplemented with 10 % FBS, and C4-2B cells, in T-Medium supplemented with 10 % FBS. All cell lines were authenticated with STR analysis (Genomics core at Michigan State University, East Lansing, MI) and shown to have markers respective for each cell line as established by ATCC and also tested for mycoplasma contamination prior to use with Venor-GeM mycoplasma detection kit (Sigma Biochemicals, St. Louis, MO).
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, Rockford, IL), and equal amounts of protein were resolved by 10 % SDSPAGE. Immunoblot was performed with antibodies against pHER2 (Y1248) (Catalog # A00318-100, GenScript, Piscataway, NJ), total HER2 (Catalog #, SC-284, Santa Cruz Biotechnology, Dallas, TX) pEGFR (Y1173) (Catalog # 4407 s, Cell Signaling, Beverly, MA), total EGFR (Catalog # 4267 s, Cell Signaling, Beverly, MA), Flotillin (Catalog # 610383, BD Biosciences, San Jose, CA), β-tubulin (Catalog # SC-5274, Santa Cruz Biotechnology, Dallas, TX), pSrc (Catalog # 2101 s, Cell Signaling Technologies, Beverly, MA), total Src (Catalog # 2109 s, Cell Signaling Technologies, Beverly, MA), Gαi2 (Catalog # SC-7276, Santa Cruz Biotechnology, Dallas, TX), pAkt (S473) (Catalog # 9271 s, Cell Signaling, Beverly, MA), Akt (Catalog # 2938 s, Cell Signaling, Beverly, MA), and GAPDH (Catalog #SC-25778, 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).
Cell fractionation
A successive detergent solubilization method for isolating lipid rafts was previously described [
3] and detergent free preparation of cell lysate and density gradient centrifugation was followed as per Macdonald et al. [
31].
Invasion assay
For cells to be treated with Dasatinib (Catalog # D3307, LC Laboratories, Woburn, MA), C4-2B cells were plated on the upper chamber of Matrigel-coated transwell filters (2 × 105 cells/filter) in growth media, supplemented with 1 % FBS, containing 0.5 μM Dasatinib or vehicle control. For Pertussis toxin (PTX) (Catalog # 516561, Calbiochem, La Jolla, CA) studies, cells were pretreated with 200 ng/ml PTX for 3 h prior to cell invasion studies. For cells to be treated with Src siRNA, C4-2B cells were transfected with scrambled or Src siRNA using Lipofectamine 2000 (Invitrogen) 24 h prior to seeding 2 × 105 cells in serum free medium on Matrigel coated inserts. For both Dasatinib and siRNA conditions, cells were allowed to invade for 24 h in the presence or absence of 200 ng/mL CXCL12 added to the bottom chamber. Cotton swabs were used to remove non-migrated cells from the upper chamber, and inserts were stained with 0.9 % crystal violet. Total number of migrated cells was counted under 10X magnification in five fields. Assays were performed in triplicate. *: p < 0.05; **: p ≤ 0.005. For protein analysis, cells were treated with Dasatinib or Src siRNA as performed for the invasion assays, in the presence or absence of CXCL12. Cell lysate was collected after 24 h and analyzed by Western blot.
In vivo studies and tumor tissue analyses
PC-3 M-luc2 cells were injected intratibially on Day 0 and saline control or plerixafor treatment was started via an osmotic pump (Alzet, Cupertino, CA). Plerixafor is obtained from Genzyme Corporation and administered in the animal model using an osmotic pump at the rate of 0.5 μl per hour at 20 mg/ml concentration. After 21 or 23 days, tumor growth was determined by in vivo luciferase imaging. For treating of established tumors, mice were sacrificed and ex vivo x-ray imaging of tumor-bearing tibiae was performed at 22 or 24 days post-injection. For treating established tumors, tumors were imaged at day 17, and, based on luciferase signals, tumors were randomly divided into two groups: plerixafor vs saline (control); plerixafor or saline pumps were then implanted at 18 days post tumor cell implantation in tibiae. Further luciferase imaging was performed at day 21 to monitor tumor growth. Mice were sacrificed and ex vivo x-ray imaging of tumor-bearing tibiae was performed at 26 days post-injection. C4-2B cells were infected with lentiviruses expressing luciferase to generate C4-2B-luc cells and stable cells were selected with puromycin treatment. For gefitinib study animals imaged at 15 days post tumor cell injection were randomized as control and treatment groups. Gefitinib (Catalog # G-4408, LC Laboratories, Woburn, MA) is formulated in 0.5 % Tween 80 and administered through oral gavage at 200 mg/kg body weight. Animals were imaged at 23, 29 and 40 days and x-rays, obtained at the 40th day. Luciferase imaging was performed with either Kodak in vivo imager or Carestream in vivo Xtreme imager (Bruker, Bellerica, MA).
Immunohistochemistry
Formalin-fixed, paraffin-embedded serial tissue sections from control or plerixafor treated bone tumors were deparaffinized with xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by incubating in 3 % H
2O
2 for 20 min; 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, Burlingame, CA). Slides were incubated at 4 °C overnight in a humidified chamber with antibodies directed against CXCR4 (R&D Systems, Minneapolis, MN) or Ki67 (BD Biosciences, San Jose, CA). After washing, sections were incubated with ABC Vectastain Kit, according to manufacturer’s protocol, followed by incubation with 3, 3-diaminobenzidine tetrahydrochloride (Vector Laboratories, Inc., Burlingame, CA). Nuclei were counterstained with Mayer’s hematoxylin (Sigma-Aldrich, St. Louis, MO). Sections were then dehydrated with graded EtOH, washed with xylene, and mounted with Permount (Sigma-Aldrich, St. Louis, MO). Hematoxylin and eosin staining was also performed on bone tumor sections, and histomorphometric analyses were performed as previously described [
3] to determine tumor burden, cortical bone area, and trabecular bone area.
Statistical analyses
Data were analyzed using GraphPad Prism software and Microsoft Excel 2008. All data are presented as mean ± SE. The in vivo luciferase imaging was performed with two different machines (Kodak invivo imaging (old) and Bruker in vivo Xtream (new) equipment) in case of Figs.
4b, c and
5b , c. Therefore, the expression levels of photons generated by the old machine were normalized by those by the new machine using their geometric means as follows. Suppose there are n and m photon expression levels generated by old and new machines, respectively (i.e., X
1,
x
2, …,
x
n
;
y
1,
y
2, …,
y
m
). Then the
i th expression level
x
i
for the old machine will be normalized by
\( {\overline{x}}_i={x}_i\cdot {\left({\prod}_{k=1}^m{y}_k\right)}^{\frac{1}{m}}/{\left({\prod}_{j=1}^n{x}_j\right)}^{\frac{1}{n}}, \) where
i = 1, 2, …,
m.
Statistical comparisons were performed using Wilcoxon rank sum test and a p-value < 0.05 was considered statistically significant.
Discussion
The CXCL12/CXCR4 axis has been shown to be involved in metastasis of several types of cancers, including prostate cancers. In support of CXCL12/CXCR4 function in tumor metastasis, herein, we show that: i) CXCL12/CXCR4 transactivates members of growth factor receptors, EGFR and HER2 and that this transactivation is largely confined to lipid raft membrane microdomains in PC cells; ii) Gαi protein activation is required for downstream signaling involving HER2 and Src, and constitutively active Gαi proteins can activate HER2 and Src signaling, independent of CXCR4 activation; iii) Src activation mediates CXCL12/CXCR4 induced cell invasion; iv) plerixafor is effective in inhibition of tumor growth, when given at the time of tumor implantation, but not effective against established tumors, suggesting the CXCL12/CXCR4 axis is crucial for initial interaction with bone microenvironment and that this interaction dictates subsequent growth of bone tumors; and v) the pan EGFR family member inhibitor gefitinib inhibited growth of established bone tumors, suggesting that growth factor signaling is critical for the expansion/enlargement of bone metastases.
Lipid rafts are specialized entities in plasma membrane, enriched with tightly packed saturated lipids and cholesterol which gives liquid ordered states. These raft microdomains are known to segregate different constituents on the membrane thereby facilitating signal transduction. Previous studies show that CXCR4 signaling localizes to lipid raft membrane microdomains and that disruption of lipid rafts leads to inhibition of basal and CXCL12 signaling [
3,
6]. The association of CXCR4 with lipid rafts in hematopoietic stem cells was shown to promote bone marrow retention and regulate homing as well as mobilization of hematopoietic stem cells [
39]. Previous studies used detergents to isolate lipid rafts based on the fact that these rafts were insoluble in non-ionic detergents, but the use of detergents was implicated in artificial coalescence of signaling proteins. To address this issue we used detergent-free cell lysate preparations for lipid raft isolation using the sucrose density buoyant centrifugation method and show that the lipid raft marker flotillin co-sediments with CXCR4 and phosphorylated HER2 and Src kinases (Fig.
1d). Previous studies also show that EGFR family members localize to lipid raft preparations in detergent-free conditions [
33,
35]. These observations suggest that CXCR4 and its signaling partners localize to lipid rafts and initiate signaling events leading to cell invasion.
G-proteins are key regulators of G-protein coupled receptor (GPCR) signaling and were shown to promote oncogenic signaling. Previous studies show that activated forms of G12 and G13 promote PC cell invasion, and G
αi proteins were also shown to promote cell migration. Our data show that PTX abrogated both HER2 and Src phosphorylation and cellular invasion, suggesting that G
αi proteins are indispensable for CXCR4 induced cellular invasion. Moreover, we show that constitutively activated G
αi protiens are sufficient to mediate HER2 and Src phosphorylation and this phosphorylation may contribute to cellular invasion of PC cells, suggesting that CXCR4 activated G
αi proteins are sufficient for PC cell invasion. Recent studies support the role of G
αi proteins in Src induced formation of invadosomes, which are cellular protrusions exhibited in migrating/invading cells, where a transient activation of Cdc42 is implicated downstream of GPCR activation [
40]; thus, we cannot rule out the role of Cdc42 downstream of CXCR4 activated Src activation in PC cells.
CXCL12/CXCR4 signaling regulates hematopoietic stem cell (HSC) movement in and out of bone marrow. HSCs home to the CXCL12 rich endosteal niche in bone marrow, where upon entering the bone, they anchor to the niche through CXCR4 activation of α4β1 integrins with vascular cell adhesion molecule 1 (VCAM1) expressed in the niche. These chemokine (CXCL12/CXCR4) and cell adhesion molecule (VCAM1/α4β1) interactions restrain the HSCs in the bone marrow niche. Plerixafor is a stem cell mobilizer, where it competitively inhibits CXCL12 binding to CXCR4, thereby disrupting the HSC interaction with bone marrow niches, leading to exit of cells from bone marrow. Here we show that inhibition of CXCR4 with plerixafor is effective against bone tumor growth when given initially during tumor implantation. Similar results were previously documented for neutralization of CXCR4 with anti-CXCR4 antibodies in an intracardiac model of metastases where anti-CXCR4 antibodies reduced total metastatic burden, including that of long bone [
29]. Recent studies suggest that initial arrival of tumor cells in the bone microenvironment leads to competition of tumor cells with HSCs for occupation at osteoblastic niches [
2]. Since both HSCs and PC cells use the CXCL12/CXCR4 axis for occupying the osteoblastic niche, plerixafor inhibited CXCL12/CXCR4 interactions, leading to reduced tumor growth when given at the time of tumor implantation. Reduction in tumor growth was also accompanied by reduced tumor induced bone destruction. This suggests that plerixafor not only mediated egress of HSCs from bone but also inhibited initial interaction of tumor cells with the osteoblastic niche, leading to inhibition of tumor growth. In support of our observation, while this manuscript was in preparation, Gravina et al. showed that plerixafor inhibited intratibial tumor growth when administered right after tumor cell implantation [
41]. As the putative PC stem cells use the CXCR4 pathway for metastasis and chemotherapies often are effective in eliminating these cell populations [
42], plerixafor mediated inhibition of CXCR4 may inhibit bone metastasis by these cell populations.
Plerixafor administration to established tumors did not significantly impact tumor growth, suggesting that, at high tumor burden, plerixafor mediated egress of tumor cells from bone, as suggested by previous studies [
2], is overcome by the growth signals in tumors. CXCL12/CXCR4 transactivates members of growth factor receptor in PC cells [
3,
6], and expression of HER2 in PC patients correlates with tumor cell proliferation [
43] and activates androgen receptor signaling in advanced disease [
44]. To address the potential role of growth factor receptor activation contributing to the bone tumor growth, we treated established bone tumors with gefitinib and found our data (Fig.
6) support the notion that inhibition of growth factor receptor mediated signals with gefitinib led to reduction in tumor burden and tumor induced osteolysis in bone tumors. It remains to be determined that combination of the two therapies have an added effect over individual therapies, but based on the mechanism of target inhibition combination therapy may provide a more efficacious inhibition of bone tumor growth.
Taken together, CXCL12/CXCR4 signaling has a dual impact in bone metastasis: newly arrived cancer cells in bone use CXCL12/CXCR4 and integrin interactions to localize to the endosteal niche, and established bone tumors use CXCL12/CXCR4 transactivated growth factor receptor signaling for expansion of bone tumors.
Conclusions
CXCL12/CXCR4 transactivation of members of growth factor receptors exclusively occurs in lipid raft membrane microdomains. Gαi protein activation is required for downstream signaling involving EGFR, HER2 and Src in lipid raft membrane microdomains. Plerixafor, a competitive CXCR4 inhibitor and a stem cell mobilizer, is effective in inhibiting initial establishment of tumor cells into the bone microenvironment, whereas the same drug is ineffective in containing the expansion of pre-existing bone metastasis. Interestingly, in our model system, the growth factor receptor inhibitor gefitinib is highly effective against expanding bone tumors. Based on our preclinical observations, plerixafor may be a candidate drug for the patient populations which are high risk for developing metastasis, with low metastasis burden, and who are prone to relapse, whereas gefitinib may be a candidate for patients with metastatic disease with rising PSA. Further experiments are in progress to determine the efficacy of different drug regimens (sequence of gefitinib and plerixafor) with or without chemotherapy combination for treating established bone tumors.
Acknowledgements
We would like to thank Genzyme Corporation for gifting plerixafor. We would like to thank Dr. Diego Sbrissa for critically reading the manuscript.