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Adiponectin is widely known as an adipocytokine with therapeutic potential for its markedly protective function in the pathogenesis of obesity-related disorders, metabolic syndrome, systemic insulin resistance, cardiovascular disease and more recently carcinogenesis. In the present study, we show that adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Further analysis of the underlying molecular mechanisms revealed that adiponectin treatment increased AMP-activated protein kinase (AMPK) phosphorylation and activity as evident by increased phosphorylation of downstream target of AMPK, acetyl-coenzyme A carboxylase and inhibition of p70S6 kinase (S6K). Intriguingly, we discovered that adiponectin treatment increases the expression of tumor suppressor gene LKB1 in breast cancer cells. Overexpression of LKB1 in breast cancer cells further increased adiponectin-mediated phosphorylation of AMPK. Using isogenic LKB1 knockdown cell line pair, we found that LKB1 is required for adiponectin-mediated modulation of AMPK–S6K axis and more importantly, inhibition of adhesion, migration and invasion of breast cancer cells. Taken together these data present a novel mechanism involving specific upregulation of tumor suppressor gene LKB1 by which adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Our findings indicate the possibility of using adiponectin analogues to inhibit invasion and migration of breast cancer cells.
The prevalence of obesity in the developed world has increased greatly in recent years (Calle et al., 2003; Rose et al., 2004)and is an independent risk factor for the development of breast cancer. Recently, a study examining the relationship of obesity with mortality from breast cancer found that obese women in the highest quintile of body mass index have double the death rate from breast cancer when compared with women in the lowest quintile (Daling et al., 2001; Petrelli et al., 2002). With epithelial and other cells accounting for only approximately 10% of human breast volume, adipocytes make up the bulk of the human breast. In many human breast cancers, there is reduced connective tissue separating the adipocytes from tumor cells. Also, carcinomas invade through the basement membrane and infiltrate fibrous tissue barriers, resulting in close positioning that allows increased paracrine interactions between the adipocytes and breast epithelial cells that might directly affect breast tumorigenesis (Schaffier et al., 2007). Adipocytes produce various hormones, cytokines and growth factors, collectively called adipocytokines (Vona-Davis and Rose, 2007).
Adiponectin (also known as ACRP30, apM1, adipoQ and GBP28)(Scherer et al., 1995; Hu et al., 1996; Maeda et al., 1996; Nakano et al., 1996)is an important adipocytokine that is considered a ‘guardian angel adipocytokine’ for its protective function against obesity- related disorders and the metabolic syndrome, particularly in the pathogenesis of type II diabetes and cardiovascular disease (Spranger et al., 2003; Matsuzawa et al., 2004; Maahs et al., 2005). Adiponectin has been shown to suppress proliferation and activation of immune cells (Fantuzzi, 2005), downregulate vascular adhesion molecules in endothelial cells and inhibit smooth muscle migration (Goldstein and Scalia, 2004). Adiponectin can directly bind certain growth factors to control their bioavailability (Wang et al., 2005). Recent research has expanded to establish a function for adiponectin in cancer (Kelesidis et al., 2006). AdipoR1 and AdipoR2 (Yamauchi et al., 2003a)and T-cadherin (Hug et al., 2004)have been identified as adiponectin receptors that mediate the cellular functions of adiponectin in a tissue-dependent manner (Chen et al., 2005). Importantly, epidemiological studies have linked low levels of plasma adiponectin associated with obesity with many common forms of cancer (Barb et al., 2006; Kelesidis et al., 2006). In several independent studies, an association of low-serum adiponectin levels has been linked with increased risk of breast cancer in both postmenopausal and premenopausal women (Mantzoros et al., 2004; Chen et al., 2006), independent of age, menopause status, hormone receptor status, lymph node metastasis, status of ER and Her2/neu (Chen et al., 2006). Most importantly, some studies have suggested that breast tumors arising in women with low-serum adiponectin levels may have a more aggressive phenotype (large size of tumor, high histological grade and increased metastasis)(Miyoshi et al., 2003; Mantzoros et al., 2004; Chen et al., 2006). Several recent studies have shown that adiponectin mediates anti-proliferative response in human breast cancer cells (Dieudonne et al., 2006; Wang et al., 2006; Arditi et al., 2007; Takahata et al., 2007; Dos Santos et al., 2008; Grossmann et al., 2008). Analysis of adiponectin expression in breast tumor samples and adjacent tissues (Jarde et al., 2008)suggests that adiponectin secreted from mammary adipocytes might affect malignant properties of breast cancer cells in the breast tumor microenvironment by paracrine interactions. In the present study we specifically investigated the effect of adiponectin on the malignant properties of breast cancer cells, including adhesion, migration and invasion and also examined the underlying molecular mechanisms. Intriguingly, we discovered that adiponectin increases the expression of tumor suppressor gene LKB1 to modulate the signaling pathway involving AMPK–S6K axis. We directly tested the requirement of LKB1 in adiponectin-mediated inhibition of malignant properties of breast cancer cells. Our results showed that LKB1 is required for adiponectin-mediated modulation of AMPK–S6K axis and inhibition of adhesion, migration and invasion of breast cancer cells.
Epidemiological studies have shown that low adiponectin levels are significantly associated with an increased breast tumor growth and metastasis (Miyoshi et al., 2003; Mantzoros et al., 2004; Barb et al., 2006; Chen et al., 2006; Kelesidis et al., 2006). Two important steps in the metastatic process involve the ability of cancer cells to adhere to extracellular matrix (ECM) components and subsequently invade and migrate (Steeg and Theodorescu, 2008). We examined the effect of adiponectin on breast cancer cell adhesion to ECM, invasion and migration using in vitro adhesion, scratch migration, electric cell-substrate impedance sensing (ECIS)migration and Matrigel invasion assays. Adiponectin treatment resulted in inhibition of migration of breast cancer cells (Figure 1a)in comparison to untreated cells. Next, we performed a quantitative real-time impedance assay using an ECIS-based technique (Saxena et al., 2008)to follow migration of MCF7 and T47D breast cancer cells. Control untreated cells showed increase in resistance showing increased migration to reach the resistance values of the non-wounded cells at the start of the experiment whereas adiponectin-treated cells showed decrease in resistance showing decreased migration. Notably, adiponectin-treated cells never reached the values of non-wounded cells showing significant inhibition of migration potential (Figure 1b). We also examined the growth-inhibitory effects of adiponectin on breast cancer cells using a long-term colony formation assay. We found that adiponectin treatment significantly decreased colony size and number (Figure 1c). Cell adhesion assays were performed using fibronectin as an adhesion substrate. Adiponectin-treated breast cancer cells showed a significant reduced adhesion to fibronectin as compared to untreated controls (Figure 1d). Adiponectin treatment also effectively inhibited invasion of breast cancer cells through Matrigel (Figure 1e). Collectively, these results showed that adiponectin inhibits adhesion, invasion and migration of breast cancer cells.
Recent studies have shown that adiponectin stimulates multiple pathways (nuclear factor (NF)-κB, peroxisome proliferator-activated receptor (PPAR)-á, p38 mitogen-activated protein (MAP)kinase and AMPK)in a target tissue-dependent manner (Yamauchi et al., 2002; Cacicedo et al., 2004; Luo et al., 2005a; Yoon et al., 2006). We investigated the involvement of NF-κB, PPAR-α, p38MAPK and AMPK in breast cancer cells in response to adiponectin and found that adiponectin only increases the activity of AMPKα1 (AMPK)in breast cancer cells. No changes were observed for NF-κB, PPAR-α, p38MAPK and AMPKα2 (data not shown). Adiponectin stimulated phosphorylation of AMPK at Thr172 in MCF7 and T47D cells within 15 min after treatment with a significant increase after 2 h of treatment. Adiponectin treatment had no effect on total AMPK protein expression levels (Figure 2a). Given the function of the yeast homologue of AMPK (SNF1)in regulating gene expression (Hardie, 2005), we examined whether AMPK might be localized in the nucleus in response to adiponectin treatment. Immunofluorescence analysis showed that adiponectin treatment increased nuclear accumulation of phosphorylated AMPK (Figure 2a). AMPK phosphorylation at Thr172 has been widely associated with its activation. Once activated, AMPK directly phosphorylates and inactivates a number of ATP-consuming metabolic enzymes including acetyl-coenzyme A carboxylase (ACC)(Hardie, 2004; Motoshima et al., 2006). We examined the phosphorylation of ACC to evaluate AMPK activity upon adiponectin treatment. Adiponectin treatment led to increased phosphorylation of ACC in MCF7 and T47D cells within 15 min as compared to untreated cells. Maximal levels of ACC phosphorylation were observed at 1–2 h after adiponectin treatment (Figure 2c). Immunofluorescence analysis of MCF7 and T47D cells treated with adiponectin for 2 h showed increased nuclear accumulation of phosphorylated ACC (pACC)(Figure 2d)as compared to untreated cells. Control experiments with secondary antibody (results not shown)gave a very faint background fluorescence that was distributed uniformly throughout the cells irrespective of the treatment.
Activation of AMPK suppresses mammalian target of rapamycin (mTOR)signaling and the molecular mechanism involves phosphorylation of tuberous sclerosis complex protein TSC2 at Thr1227 and Ser1345 that increases the activity of TSC1–TSC2 complex to inhibit mTOR (Hardie, 2004; Luo et al., 2005b). The most characterized downstream effectors of mTOR are the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1 or S6K)and the eukaryotic translation initiation factor 4E (elF4E)-binding protein (4E-BP1). Phosphorylation of S6K has been widely used to assess changes in mTOR activity in response to various growth factor pathways (Martin and Hall, 2005). We next examined the effect of adiponectin on mTOR activity in MCF7 and T47D cells. Adiponectin decreased phosphorylation of S6K within 15–30 min after treatment and resulted in significant inhibition of S6K phosphorylation within 4 h after treatment whereas no change was observed in the expression of total S6K protein (Figure 3a). S6K translocate to discrete cellular compartments through nucleocytoplasmic shuttling to regulate various processes such as the regulation of protein synthesis, cytoskeleton, cell survival, RNA processing and transcription (Ruvinsky and Meyuhas, 2006). Immunofluorescence analysis of adiponectin-treated MCF7 and T47D cells exhibited reduced expression of phosphorylated S6K (pS6K) protein both in nucleus and in cytoplasm as compared to untreated cells (Figure 3b). Control experiments with secondary antibody (results not shown)gave an extremely faint background fluorescence that was distributed uniformly throughout the cells irrespective of the treatment. Together, these data suggest that adiponectin signaling involves increased AMPK activity as well as inhibition of mTOR activity in breast cancer cells.
The tumor suppressor LKB1 (also known as Stk11)is an evolutionarily conserved serine/threonine protein kinase that has a broad range of cellular functions including tumor suppression, cell polarity, cell-cycle regulation and promotion of apoptosis (Hardie, 2005). LKB1 has recently been identified as a critical upstream kinase for AMPK regulating its activity. Intriguingly, we found that adiponectin increases expression of tumor suppressor LKB1 in MCF7 and T47D cells in a temporal manner with maximal expression at 2 h (Figure 4a). Human LKB1 is both nuclear and cytoplasmic but a mutant of LKB1 lacking the nuclear localization signal still retains ability to suppress cell growth, suggesting that the cytosolic pool of LKB1 has an important function in mediating its tumor suppressor properties (Tiainen et al., 2002; Alessi et al., 2006). Also, upon activation with pseudokinase STRAD binding, LKB1 gets transported to cytoplasm and functions in cell growth regulation and generation of cell polarity (Hardie, 2005). Immunofluorescence analysis of adiponectin-treated MCF7 and T47D cells revealed that adiponectin treatment increases cytoplasmic accumulation of LKB1 as compared to untreated cells (Figure 4b). Control experiments with secondary antibody (results not shown)gave an extremely faint background fluorescence that was distributed uniformly throughout the cells irrespective of the treatment. Recently, LKB1 and Ca(2+)/calmodulin-dependent protein kinase kinases have been identified as upstream kinases for AMPK (Witters et al., 2006). Overexpression of Flag-tagged LKB1 in MCF7 and T47D cells resulted in increased phosphorylation of AMPK and reduced phosphorylation of S6K whereas no change was observed in the expression of AMPK and S6K proteins (Figure 4c). We further investigated the involvement of LKB1 in adiponectin-mediated increased AMPK phosphorylation in breast cancer cells using LKB1 overexpression constructs. Adiponectin treatment significantly increased phosphorylation of AMPK in a time-dependent manner as compared to untreated cells (Figure 4d). Interestingly, LKB1 overexpression increased AMPK phosphorylation in breast cancer cells, which was significantly increased in response to adiponectin treatment. Adiponectin treatment in LKB1 overexpressing cells resulted in approximately fourfold increase in AMPK phosphorylation as compared to vector-transfected, untreated cells (Figure 4d).
Variable expression of LKB1 in breast cancer cells has been reported. Shao and group (Shen et al., 2002) showed that their MDA-MB-231 cells lack LKB1 whereas a more recent study (Phoenix et al., 2009) showed the presence of LKB1 mRNA and protein in MDA-MB-231 cells using western blot analysis and quantitative RT-PCR. To unequivocally determine the expression of LKB1 in MDA-MB-231 cells, we procured MDA-MB-231 cells from two different labs (Dr Nancy Davidson, Director, University of Pittsburgh Cancer Institute and Dr Lily Yang, Associate Professor, Hematology and Medical Oncology, Emory University) and ATCC (Manassas, VA, USA). We analysed the presence of LKB1 in these cell lines and our data clearly showed the presence of LKB1 in these breast cancer cells (Supplementary Figure 1a). Furthermore, treatment of MDA-MB-231 cells leads to increased phosphorylation of AMPK within 15 min after treatment (Supplementary Figure 1b). Importantly, we also found that silencing LKB1 in MDA-MB-231 cells inhibits adiponectin-induced activation of AMPK (Supplementary Figure 1c). In addition, we also found that adiponectin treatment inhibits migration and invasion of MDA-MD-231 cells using quantitative ECIS-based migration and invasion assay (Supplementary Figure 2). These results collectively show that adiponectin upregulates the expression of tumor suppressor LKB1 and LKB1 overexpression is involved in adiponectin function in breast cancer cells.
Inhibition of LKB1 abrogates adiponectin-mediated modulation of AMPK–S6K axis and inhibition of growth, adhesion and migration of breast cancer cells. To directly examine the function of LKB1 in adiponectin- mediated modulation of AMPK–S6K axis in breast cancer cells, we used LKB1shRNA lentivirus and puromycin to select for stable pools of MCF7 cells with LKB1 depletion. We analysed pLKO.1 and LKB1shRNA stable MCF7 cell pools for LKB1 protein expression by immunoblot and immunofluorescence analysis, and found that LKB1 protein expression was significantly reduced in LKB1shRNA cells as compared to pLKO.1 control cells (Figure 5a). Depletion of LKB1 in LKB1shRNA cells resulted in decreased phosphorylation of AMPK whereas total AMPK protein levels remain unchanged in both LKB1shRNA and pLKO.1 cells. We observed significant increase in S6K phosphorylation in LKB1shRNA cells using immunoblot and immunofluorescence analysis in comparison to pLKO.1 cells indicating that depletion of LKB1 increases mTOR activity. Depletion of LKB1 did not change the level of total S6K protein (Figure 5a).
We next sought to determine the biological importance of the depletion of LKB1 in the context of effect of adiponectin on AMPK–S6K axis and malignant properties of breast cancer cells. pLKO.1 and LKB1shRNA cells were treated with adiponectin and phosphorylation of AMPK and S6K was determined using western blot and immunofluorescence analysis. We found that adiponectin increased phosphorylation of AMPK and inhibited phosphorylation of S6K in pLKO.1 cells. Intriguingly, displaying a crucial function of LKB1, adiponectin treatment did not change the phosphorylation levels of either AMPK or S6K in LKB1shRNA cells (Figure 5b). Adhesion, invasion and migration are the key biological features of malignant behavior of carcinoma cells. In addition to examining the effect of LKB1 depletion on adiponectin-induced modulation of AMPK–S6K axis, we also examined the requirement of LKB1 in adiponectin-mediated inhibition of metastatic properties of breast cancer cells. As evident from Figure 6a, adiponectin treatment efficiently inhibited migration of pLKO.1 cells whereas untreated pLKO.1 cells migrated to close the wound. Our results showed that LKB1shRNA cells exhibited increased migration in the absence of adiponectin treatment. Interestingly, adiponectin treatment did not inhibit the migration of LKB1shRNA cells (Figure 6a). Next, we performed a quantitative real-time impedance assay using an ECIS-based technique to follow migration of LKO.1 and LKB1shRNA cells. LKO.1 cells treated with adiponectin displayed a decrease in resistance, showing decreased migration whereas untreated cells rapidly increase to reach the resistance values of the non-wounded cells at the start of the experiment. In a striking contrast, LKB1shRNA cells untreated and treated with adiponectin showed a similar increase in resistance exhibiting increased migration (Figure 6b). We further found that adiponectin inhibited the number and size of colonies of pLKO.1 cells in long-term colony formation assay reducing the growth of pLKO.1 cells as expected. On the other hand, adiponectin treatment did not affect colony formation of LKB1shRNA cells (Figure 6c). In addition, adiponectin inhibited adhesion of pLKO.1 cells to adhesion substrate whereas LKB1shRNA cells remained unaffected by adiponectin treatment (Figure 6d). We next examined the effect of adiponectin on invasion potential of pLKO.1 and LKB1shRNA cells and found that adiponectin inhibited invasion of pLKO.1 cells whereas LKB1shRNA cells were not affected by adiponectin treatment (Figure 6e). These results collectively show that adiponectin-induced LKB1 overexpression is indeed a crucial component of the signaling machinery used by adiponectin in modulating AMPK–S6K axis and inhibiting metastatic properties of breast cancer cells.
The following novel findings are described in this study: (1)adiponec tin treatment inhibits malignant properties such as adhesion, invasion and migration of breast cancer cells; (2)adiponect in stimulates AMPK phosphorylation and activity while reducing mTOR activity as evidenced by reduced phosphorylation of S6K; (3)ad iponectin treatment led to increased expression of tumor suppressor LKB1; (4)overexpression of LKB1 along with adiponectin treatment led to further increase in AMPK phosphorylation and (5) tumor suppressor LKB1 is required for adiponectin-mediated modulation of AMPK–S6K axis and inhibition of adhesion, invasion and migration of breast cancer cells. These results show that adiponectin treatment significantly inhibits malignant properties of breast cancer cells through modulation of LKB1– AMPK–S6K axis thus using adiponectin analogues may be a suitable therapeutic strategy for metastatic breast cancer.
Adiponectin has been previously shown to exert antiproliferative effect on breast cancer cells (Wang et al., 2006; Arditi et al., 2007; Takahata et al., 2007)by modulating glycogen synthase kinase-3β/β-catenin pathway (Wang et al., 2006)and Wnt-inhibitory factor-1 (Liu et al., 2008). Earlier studies (Korner et al., 2007; Nakayama et al., 2008)have shown that adiponectin does not induce apoptosis in T47D cells whereas MCF7 cells show a slight (Dieudonne et al., 2006)or no apoptotic response (Arditi et al., 2007). We found that adiponectin does not induce apoptosis in T47D breast cancer cells whereas MCF7 cells show slight increase in apoptosis (Supplementary Figure 3). Our studies show for the first time that adiponectin not only inhibits breast cancer cell adhesion but importantly it also inhibits properties such as invasion and migration by increasing expression of tumor suppressor LKB1. LKB1 kinase was first identified as a tumor suppressor and a key determinant in Peutz–Jeghers syndrome, an inherited propensity to gastrointestinal and other cancers including lung, pancreatic and breast cancer (Jenne et al., 1998; Hardie, 2005). LKB1 gene inactivation has been shown in a subset of sporadic lung and pancreatic cancer. Extensive analysis of LKB1 expression in breast tumors using IHC recently showed that abrogation of LKB1 expression is not common in human breast carcinoma but importantly correlates with high-grade ductal carcinoma in situ (DCIS)and high-grade invasive ductal carcinoma (Fenton et al., 2006). Of note is the fact that LKB1 expression was abrogated only in the DCIS associated with invasion and not pure DCIS cases, indicating that loss of LKB1 could potentially promote invasion. Consistent with these findings, low LKB1 protein levels have been shown to correlate with poor prognosis in breast carcinoma (Shen et al., 2002). LKB1 is involved in multiple cellular functions such as regulation of cell polarity, energy homeostasis, protein synthesis and cell-cycle arrest. Previous studies using mutant LKB1 lacking the nuclear localization signal have shown that cytoplasmic pool of LKB1 is important in mediating its tumor suppressor properties. Coexpression of LKB1 with STRAD and MO25 strikingly increased its localization in cytoplasm whereas mutant LKB1, unable to interact with STRAD and MO25, remained in nucleus (Baas et al., 2003; Boudeau et al., 2003, 2004). Adiponectin might also increase LKB1–STRAD–MO25 interaction in addition to overexpression of LKB1 thus increasing the functional pool of LKB1. LKB1 is phosphorylated on at least eight residues (Ser31, Ser325, Thr366, Ser431 by upstream kinases and autophosphorylation at Thr185, Thr189, Thr336 and Ser404). Mutation of any of these sites of phosphorylation to either Ala to abolish phosphorylation or to Glu to mimic phosphorylation has thus far not been reported to significantly affect LKB1 catalytic activity in vitro or its cellular localization (Sapkota et al., 2001, 2002; Boudeau et al., 2003; Hezel and Bardeesy, 2008). Our studies support the concept that LKB1 is important in breast cancer metastasis and increased expression of LKB1 in response to adiponectin treatment inhibits invasion and migration properties of breast cancer cells.
Upstream kinases for AMPK include LKB1 and CaMKKs. Tumor suppressor effects of LKB1 can be partly due to its ability to activate master metabolic regulator AMPK (Hardie, 2005). AMPK undergoes a conformational change in response to direct binding of AMP to its nucleotide-binding domain, exposing the activation loop of the catalytic kinase subunit. LKB1 phosphorylates a critical threonine in this activation loop to activate AMPK (Hardie, 2005). LKB1 is required for AMPK activation as genetic depletion of LKB1 in mouse embryonic fibroblasts results in a loss of AMPK activation following energy stresses that raise AMP (Hardie, 2004). We found that silencing of LKB1 in breast cancer cells resulted in decreased AMPK activation. Importantly, we also found that overexpression of LKB1 increases AMPK activation that was synergistically increased by concomitant adiponectin treatment. In addition to being directly activated by tumor suppressor LKB1, AMPK itself regulates the activation of two other tumor suppressors, TSC1 and TSC2. Heterodimeric binding partners TSC2 and TSC1 are critical regulators of Rheb that in turn regulates mTOR protein kinase that regulates a vast range of cellular activities including: transcription, translation, cell size, mRNA turnover, protein stability, ribosomal biogenesis and cytoskeletal organization (Hardie, 2004). As predicted by this molecular circuitry, cells having reduced expression of LKB1 show hyperactivation of mTOR that may contribute to carcinogenesis (Hardie, 2004, 2005). We found that LKB1 silencing resulted in increased mTOR activity whereas adiponectin-induced increased expression of LKB1 resulted in inhibition of mTOR activity in breast cancer cells. Our results suggest that adiponectin analogues may be excellent candidates as targeted therapies for carcinomas characterized by hyperactive mTOR signaling such as human familial cancer syndromes all of which are characterized by hamartoma-type tumors.
Although most of the adipocytokines are casually linked to obesity-related diseases including carcinogenesis, adiponectin has shown promising insulin-sensitizing, anti-inflammatory and antiatherogenic activities. Adiponectin levels are decreased in obesity and various obesity-related diseases. The clinical relevance of adiponectin treatment has been suggested by studies showing that treatment with adiponectin can improve glucose/lipid homeostasis, increase insulin sensitivity and prevent atherosclerosis in animal models (Berg et al., 2001; Fruebis et al., 2001; Xu et al., 2003; Yamauchi et al., 2003b; Kelesidis et al., 2006). Metformin, a first-line treatment for type II diabetes, inhibits breast cancer cell growth (Alimova et al., 2009), induces cell-cycle arrest (Zhuang and Miskimins, 2008)acting in an AMPK-dependent manner (Zakikhani et al., 2006). So far no studies have shown that metformin inhibits migration and invasion. Our studies showed that adiponectin inhibits the metastatic properties of breast cancer cells through modulation of LKB1–AMPK–S6K axis thus providing mechanistic insights for the development of adiponectin analogues. In addition to increasing adiponectin levels using adiponectin analogues, augmentation of its effectiveness by using thiazolidinediones (Swarbrick and Havel, 2008)can potentially become a future beneficial treatment for patients with breast cancer. Considering the high prevalence of obesity in the United States, our study has the potential to significantly impact the vast majority of obese patients with breast cancer by using adiponectin for inhibiting invasion and migration properties of breast tumors and improving overall survival.
Antibodies for pAMPK (phospho-AMPKα1-Thr172), AMPK (AMPKα1)and LKB1 were purchased from Cell Signaling Technology (Danvers, MA, USA). ACC and pACC (phospho-ACC)were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). S6K and pS6K (phospho-S6K-Thr389) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit Alexa Flour 488 secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA).
The human breast cancer cell lines, MCF-7 and T47D, were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA, USA)and 2 μM L-glutamine (Invitrogen). For treatment, cells were seeded at a density of 1×106 per 100mm tissue culture dish. Cultures were treated with human recombinant full-length adiponectin (BioVender, Candler, NC, USA)at 10 μg/ml for indicated durations. For ECIS migration assay, ECIS cell culture ware was purchased from Applied BioPhysics (Troy, NY, USA).
Five pre-made lentiviral LKB1 short-hairpin RNA (shRNA) constructs and a negative control construct created in the same vector system (pLKO.1)were purchased from Open Biosystems (Huntsville, AL, USA). Lentiviral helper plasmids (pCMV-dR8.2 dvpr and pCMV-VSV-G)were obtained from Addgene (Cambridge, MA, USA). Transient lentivirus stocks were prepared following the manufacturer’s protocol. One day before transfection, 1.5×106 293T cells were plated in 100 mm dishes. Cells were co-transfected with shRNA constructs (3 μg) together with 3 μg pCMV-dR8.2 dvpr and 0.3 μg pCMV-VSV-G helper constructs. After 2 days, viral stocks were harvested from the culture medium and filtered to remove nonadherent 293 T cells. To select for the breast cancer cells that were stably expressing shRNA constructs, we plated cells at subconfluent densities and infected them with a cocktail of 1 ml of virus-containing medium, 3 ml of regular medium and 8 μg/ml polybrene. Selection with 0.5–2 μg/ml of puromycin was started 48 h after lentivirus infection. After 4 weeks of selection for MCF7 cells, monolayers of stably infected pooled clones were harvested for use and cryopreserved.
Migration assays were performed following previously published protocol (Saxena et al., 2007a). For details, please see Supplementary material. All experiments were performed at least three times in triplicates.
Wound-healing assays were performed using the ECIS (Applied BioPhysics)(Saxena et al., 2008). For details please see Supplementary material. All experiments were performed at least three times in triplicates.
To perform colony formation assay (Zhong et al., 2008), we plated MCF7 and T47D breast cancer cells (single-cell suspension) in 12-well plates at a density of 250 cells per well overnight. The following day, cells were treated with 10 μg/ml human recombinant full-length adiponectin and the medium was replaced with fresh medium containing adiponectin every 3 days. After a 10-day treatment period, the medium was removed and cell colonies were stained with crystal violet (0.1%in 20% methanol). Colony numbers were assessed visually and colonies containing >50 normal-appearing cells were counted. Pictures were taken using a digital camera. All experiments were performed at least three times in triplicates.
Whole-cell lysate was prepared following previously described method (Saxena et al., 2007b). For details, please see Supplementary material. All experiments were performed at least three to five times using independent biological replicates.
MCF7, T47D, MCF7-pLKO.1 and MCF7-LKB1shRNA breast cancer cells (5×105 cells per well)were plated in four-well chamber slides (Nunc, Rochester, NY, USA)followed by treatment with 10 μg/ml human recombinant full-length adiponectin for 2 h. Fixed and immunofluorescently stained cells were imaged using a Zeiss LSM510 Meta (Zeiss, Thornwood, NY, USA)laser scanning confocal system. For detailed methodology, please see Supplementary material. All experiments were performed multiple times using independent biological replicates.
LKB1-wild-type (LKB1-WT)plasmid construct was procured from Addgene (Shaw et al., 2004). Breast cancer cells were transiently transfected with 1.0 μg of LKB1-WT plasmid using Lipofectamine 2000 (Invitrogen)according to the manufacturer’s protocol. At 48 h after transfection, the cells were treated with 10 μg/ml human recombinant full-length adiponectin for the indicated durations. Cells were harvested and total protein lysates were subjected to western blot analysis using specific antibodies as described. The experiments were performed in triplicates, and similar results were obtained from at least three independent experiments.
Cells were pretreated with 10 μg/ml adiponectin for 24 h and plated (5×104 cells per well)in 10 μg/cm2 fibronectin-coated (Sigma, St Louis, MO, USA)wells in 96-well plates followed by 60 min incubation at 37 °C (5% CO2). Adherent cells were fixed with 3% paraformaldehyde for 10 min, washed with 2% methanol for 10 min and stained with 0.5% crystal violet in 20% methanol for 10 min. Photographs were taken using a Zeiss Axioplan 2 upright microscope. The stain was eluted and absorbance at 540 nm was measured. All experiments were performed multiple times in triplicates.
For an in vitro model system for metastasis, we performed a Matrigel invasion assay by using a Matrigel invasion chamber from BD BioCoat Cellware (BD Biosciences, San Jose, CA, USA)(Saxena et al., 2008). For detailed description, see Supplementary material. All experiments were performed at least three times.
All experiments were independently performed three times in triplicates. Statistical analysis was carried out using Microsoft Excel software. Significant differences were analysed using Student’s t-test and two-tailed distribution. Data were considered to be statistically significant if P<0.05. Data are expressed as means±s.e. between triplicate experiments.
This study was supported by NIH LRP (grant to LTS), NIDDK NIH (K01DK076742 to NKS), NCI NIH (5P01CA116676-030002 to WZ), NCI NIH (R01CA131294 to DS), Wilbur and Hilda Glenn Foundation (grant to DS), CDMRP BCRP (grant BC030963 to DS), The Susan G Komen for the Cure (grant BCTR0503526 to DS), Emory University Research Council (DS), BJ Foundation (DS) and Mary K Ash Foundation (research grant to DS).
Conflict of interest
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)