Overexpression of HMGA1 promotes anoikis resistance and constitutive Akt activation in pancreatic adenocarcinoma cells

HMGA1 proteins are architectural transcription factors that are overexpressed by pancreatic adenocarcinomas. Roles of HMGA1 in mediating the malignant phenotype of this cancer are poorly understood. We tested the hypothesis that overexpression of HMGA1 promotes resistance to anoikis (apoptosis induced by anchorage deprivation) in pancreatic cancer cells. HMGA1 cDNA was stably transfected into MiaPaCa2 human pancreatic adenocarcinoma cells (which have low baseline expression levels of HMGA1). Cells were grown in suspension on PolyHEMA-coated plates and their susceptibility to anoikis was assayed using flow cytometry. Overexpression of HMGA1 was associated with marked reductions in susceptibility to anoikis in concert with increases in Akt phosphorylation (Ser473) and in Akt kinase activity and with reductions in caspase 3 activation. Inhibition of phosphoinositidyl-3 (PI3-K)/Akt pathway with either the small molecule inhibitor LY294002 or dominant-negative Akt resulted in reversal of anoikis resistance induced by HMGA1 overexpression. Further, RNA interference-mediated HMGA1 silencing in MiaPaCa2 and BxPC3 (a human pancreatic adenocarcinoma cell line with high baseline levels of HMGA1 expression) cells resulted in significant increases in susceptibility to anoikis. Our findings suggest HMGA1 promotes anoikis resistance through a PI3-K/Akt-dependent mechanism. Given the putative associations between anoikis resistance and metastatic potential, HMGA1 represents a potential therapeutic target in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is among the deadliest of all human cancers, with 5-year survival rates averaging less than 5% (Jemal et al, 2006). The propensity for pancreatic adenocarcinoma cells to metastasise early in the course of disease progression makes this cancer particularly refractory to standard therapies. Characterising the underlying mechanisms mediating metastatic dissemination in pancreatic adenocarcinoma may reveal novel targets for inhibiting this process, which is so aggressive in this cancer.
Anoikis, derived from a Greek word meaning 'homelessness', was used in describing the observation that depriving cells from attachment to matrix triggers apoptosis in these cells (Frisch and Francis, 1994). Increasingly, metastasis is conceptualised to be a multistep process facilitated by the evolution of anoikis-resistant subsets of cancer cells that are capable of surviving in the blood stream during dissemination after they detach from the primary tumour and its stroma. Indeed, numerous studies suggest a close correlation between signalling events mediating cellular anoikis resistance in vitro and those mediating metastatic potential in vivo (Yawata et al, 1998;Zhu et al, 2001;Douma et al, 2004;Berezovskaya et al, 2005).
Although these correlative data suggest a potential role for HMGA1 in cancer pathogenesis, the underlying biology of HMGA1 in the context of cancer progression is poorly understood. The purpose of this study was to test the hypothesis that HMGA1 promotes anoikis resistance in pancreatic adenocarcinoma. Our observations indicate that HMGA1 promotes anoikis resistance through the phosphoinositidyl-3 kinase (PI3-K)/Akt pathway.

Cells and cell culture
MiaPaCa2 and BxPC3 human pancreatic ductal adenocarcinoma cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in DMEM containing 10% fetal bovine serum (FBS) (Gibco Life Technologies Inc., Gaithersburg, MD, USA) and incubated in a humidified (371C, 5% CO 2 ) incubator, grown in 75-cm 2 culture flasks, and passaged on reaching 80% confluence.

Reagents and dominant-negative Akt adenovirus
The PI3-K-inhibitor LY294002 was purchased from Calbiochem (San Diego, CA, USA). Anti-HMGA1, anti-lamin B and anti-Akt antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho Akt (ser473) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Adenovirus expressing dominant-negative murine Akt1 and control virus (Ad-CMV-null) (both titred at 1 Â 10 10 PFU ml À1 ) were purchased from Vector Biolabs (Philadelphia, PA, USA). Adenoviral infection was performed at multiplicity of infection of 10 in the presence of 6 mg ml À1 polybrene for 12 h. Experiments were performed on cells 48 h following infection.

Expression vector and transfection
The HMGA1-coding sequence was PCR amplified from IMAGE clone 5399570 (GenBank accession no. BC063434) using genespecific primers modified to include the appropriate restriction sites at their 5 0 end. The primers used were: forward, 5 0 -TTTTGATATCATGAGTGAGTCGAGCTCGAAG-3 0 and backward, 5 0 -TTTTGAATTCTCACTGCTCCTCCTCCGAGGA-3 0 . Purified PCR products were digested with EcoRV and EcoRI, before ligation into a EcoRV/EcoRI-digested pIRES-puro3 vector (Clontech, Palo Alto, CA, USA). The expression plasmid was named pIRES-HMGA1. MiaPaCa2 cells were transfected with pIRES-HMGA1 or empty pIRES-puro3, which acted as a control, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. Stable clones were selected by exposure to incrementally increasing concentrations of puromycin (Invivogen, San Diego, CA, USA), isolated using cloning cylinders and maintained in medium containing 3 mg ml À1 puromycin (Invivogen). Clones pIRES-HMGA1.1 and pIRES-HMGA1.2, which expressed the highest levels of HMGA1, were used for further studies.

Anoikis induction and flow cytometry
Anoikis was assayed by plating cells on polyHEMA-coated plates. A solution of 120 mg ml À1 polyHEMA (Sigma) in 100% ethanol was made and diluted 1:10 in 95% ethanol; 0.95 ml mm À2 of this solution was overlaid onto 35-mm wells and left to dry in a heated dryer system for 12 h. Before use, wells were washed twice with PBS and once with DMEM. In all, 1 Â 10 6 cells of each line, suspended in 2 ml DMEM with 10% FBS were incubated in the polyHEMA-coated wells for 12-18 h in a humidified (371C, 5% CO 2 ) incubator. Cells were harvested and resuspended in 0.3 ml of PBS containing 2% FBS and 0.1 mM EDTA. Apoptosis staining was performed using 1 ml ml À1 YO-PRO-1 and propidium iodide (Vybrant Apoptosis Assay Kit #4; Molecular Probes, Eugene, OR, USA). Cells were incubated for 30 min on ice and then analysed by flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ, USA), measuring fluorescence emission at 530 and 575 nm. Cells stained with the green fluorescent dye YO-PRO-1 were counted as apoptotic; necrotic cells were stained with propidium iodide. The number of apoptotic cells was divided by the total number of cells (minimum of 10 4 cells), resulting in the apoptotic fraction. Data were analysed using CellQuest software (Becton Dickinson). All assays were performed in triplicates.

Western blotting
Cells were harvested and rinsed twice with PBS. Total cell extracts were prepared with lysis buffer (20 mM Tris, pH 7.5, 0.1% Triton X, 0.5% deoxycholate, 1 mM PMSF, 10 mg ml À1 , aprotinin, 10 mg ml À1 leupeptin) and cleared by centrifugation at 15 000 g, 41C. Nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents based on the manufacturer's instruction (Pierce, Rockford, IL, USA). Total protein concentration was measured using the BCA assay kit (Sigma) with bovine serum albumin as a standard, according to the manufacturer's instructions. Total cell lysates containing 50 mg total protein or nuclear protein containing 10 mg total protein were subjected to 10% SDS/PAGE and the resolved proteins transferred electrophoretically to PVDF membranes (Invitrogen). Equal protein loading was confirmed by Coomassie (BioRad, Hercules, CA, USA) staining of the gel. After blocking with PBS containing 3% bovine serum albumin for 1 h at room temperature, membranes were incubated with 3 -5 mg ml À1 antibody in PBS containing 0.1% Tween-20 overnight at 41C. Chemoluminescensce detection (Amersham Biosciences, Piscataway, NJ, USA) was performed in accordance with the manufacturer's instructions. The densitometric signal was quantified using ImagePro Plus software version 4.0 (Media Cybernetics, Silver Spring, MD, USA) and normalised to that of loading control as appropriate. Blots were performed in triplicate in at least three independent experiments. Mean densitometric values (7standard Deviation (s.d.)) are shown.

Fluorometric real-time Akt kinase assay
Assay was performed using the Omnia Lysate Akt kinase assay (Biosource-Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Briefly, total cell lysate equivalent to 5 mg ml À1 was loaded into each well. Following addition of fluorescent Akt substrates and kinase buffer, fluorescent signal was acquired real-time (excitation, 360 nm; emission, 485 nm) for 60 min using SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The Akt kinase activity was calculated from the slope of activity curve. Experiments were performed in triplicates on at least three occasions.

Fluorometric caspase profiling
Whole-cell lysates were assayed for caspases 3 activity using the BD ApoAlert Caspase Assay Plate (BD Biosciences Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions. Plates were read (excitation, 360 nm; emission, 480 nm) using SpectraMax M5 microplate reader in fluorescence mode (Molecular Devices). All measurements were performed in triplicate, each with three determinations for each condition.

Proliferation assay
Cell proliferation was quantified using an MTS (3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium) assay (CellTiter 96 Aqueous One Solution Assay, Promega, Madison, WI, USA), in accordance to the manufacturer's instructions and confirmed by cell counting. Logarithmically growing cells were seeded into 96-well plates at 5 Â 10 3 cells per well and allowed to adhere overnight in medium containing 10% FBS. Cell proliferation was determined after 48 h. Plates were read with the use of the SpectraMax M5 microplate spectrophotometer (Molecular Devices) at a wavelength of 490 nm. Ten samples were used for each experimental condition and experiments were performed in triplicate. At identical time points, cell counting was performed. Cells were trypsinised to form a single-cell suspension. Viable cells, determined by trypan blue exclusion, were counted with the use of a Neubauer hemocytometer (Hausser Scientific, Horsham, PA, USA).

Statistical analysis
Differences between groups were analysed using Student's t-test, multifactorial ANOVA of initial measurements and Mann -Whitney U-test for nonparametric data, as appropriate, using Statistica 5.5 software (StatSoft Inc., Tulsa, OK, USA). In cases in which averages were normalised to controls, the s.d. of each nominator and denominator were taken into account in calculating the final s.d.. Po0.05 was considered statistically significant.

HMGA1 overexpression promotes anoikis resistance in MiaPaCa2 pancreatic adenocarcinoma cells
Stable overexpression of HMGA1 was achieved in MiaPaCa2 cells, which have low inherent expression levels of HMGA1. Two stable HMGA1-overexpressing clones were selected and named pIRES-HMGA1.1 and pIRES-HMGA1.2. HMGA1 overexpression was confirmed on Western blot analysis (Figure 1). Levels of HMGA1 overexpression in pIRES-HMGA1.1 and pIRES-HMGA1.2 were four-and 3.5-fold higher than in control cells, respectively. There was no difference in the levels of HMGA1 expression between the empty pIRES-puro3 transfectants and parental MiaPaCa2 cells. Following 18 h of anchorage-deprivation (on polyHEMA plates), pIRES-HMGA1.1 and pIRES-HMGA1.2 clones showed significantly increased anoikis resistance (mean anoikis fractions of 11 and 13%, respectively) when compared to controls (mean anoikis fractions for parental MiaPaCa2 and empty pIRES-puro3 transfectants were 26 and 27%, respectively, Figure 2A and 2B).

HMGA1 overexpression results in protection from caspasemediated apoptosis
Given that disruption of cell -matrix interactions can trigger anoikis via caspase-dependent apoptosis, we examined the effects of HMGA1 overexpression on caspase 3 activity (a central mediator of apoptosis) in the context of anchorage deprivation. During induction of anoikis on polyHEMA plates, HMGA1overexpressing clones demonstrated markedly reduced levels of caspase 3 activity compared to parental MiaPaCa2 or pIRES-puro3 controls ( Figure 2C).
Overexpression of HMGA1 increases levels of Akt phosphorylation and Akt kinase activity PI3-K/Akt-signalling pathway is of critical importance in mediating anoikis resistance and enhancing anchorage-independent cell cycle progression (Moore et al, 1998;Nguyen et al, 2002). Given these observations, we sought to determine if HMGA1 overexpression would modulate Akt phosphorylation at Ser473, a marker of Akt activation. HMGA1 overexpression resulted in elevated Akt (Ser473) phosphorylation ( Figure 3A). Consistent with this finding, HMGA1 overexpression was found to be associated with increases in Akt kinase activity ( Figure 3B).

Inhibition of Pl3-K/Akt signalling reverses HMGA1 overexpression-induced anoikis resistance
Having demonstrated that HMGA1 overexpression induces constitutive activation of PI3-K/Akt pathway, we next examined the effects of inhibiting this pathway in cells overexpressing HMGA1. First, we used a specific small molecule inhibitor of PI3-K, LY294002, to assess the effects of inhibiting PI3-K on anoikis resistance of these cells. Addition of 25 mM of LY294002 significantly increased anoikis fractions in pIRES-HMGA1.1 and pIRES-HMGA1.2 clones ( Figure 3C). Treatment of parental MiaPaCa2 and empty pIRES-puro3 controls with LY294002 also inhibited anoikis resistance, although to lesser extent than for pIRES-HMGA1.1 and pIRES-HMGA1.2 clones. Second, infection of pIRES-HMGA1.1 and pIRES-HMGA1.2 clones with adenovirus expressing a dominant-negative Akt construct also significantly increased anoikis fractions in both HMGA1-overexpressing clones ( Figure 3D). Parental MiaPaCa2 and empty pIRES-puro3 controls also exhibited increased anoikis fractions with infection of adenovirus expressing dominant-negative Akt construct, but to a lesser extent than the HMGA1-overexpressing clones. Taken together, these results suggest that HMGA1 overexpressioninduced anoikis resistance is dependent on PI3-K/Akt signalling.

Targeted RNAi of HMGA1 in MiaPaCa2 and BxPC3 pancreatic adenocarcinoma cells increases susceptibility to anoikis
Given the effects of HMGA1 overexpression on anoikis resistance in MiaPaCa2 cells, we asked if silencing of HMGA1 in the same cell line will have the reverse effects. In addition, we also selected BxPC3 pancreatic adenocarcinoma cell line for RNAi experiments as these cells have high levels of HMGA1 expression at baseline. In these lentivirus-mediated RNAi experiments, we developed pooled stable transfectants. Stable transfectants derived from infection with lentivirus developed from empty PLKO.1 and scramble shRNA transfer plasmids served as controls. We achieved at least 90% silencing of HMGA1 expression in both MiaPaCa2 and BxPC3 cells ( Figure 4A). Of note, neither HMGA1 overexpression nor silencing had any impact on lamin B expression (used to normalise for protein loading, Figures 1 and 4A). HMGA1 silencing was associated with significant increases in anoikis fractions in both cell lines ( Figure 4B). Modulation of HMGA1 expression did not have an impact on growth characteristics of pancreatic adenocarcinoma cells Neither overexpression ( Figure 5A) nor targeted suppression of HMGA1 expression ( Figure 5B) had any impact on proliferation rates of pancreatic adenocarcinoma cells studied.

DISCUSSION
Pancreatic adenocarcinoma is associated with dismal prognosis, in large part resulting from metastatic disease, even in patients initially deemed to be surgically resectable. We have focused our efforts in studying the mechanisms underlying metastasis in   pancreatic adenocarcinoma, with the goal of rationally identifying molecular targets that can be exploited to inhibit this process. In our work, we have used anoikis resistance as an in vitro correlate of metastatic potential. In this study, we have shown that HMGA1 overexpression confers anoikis resistance to pancreatic adenocarcinoma cells by activating the prosurvival PI3-K/Akt signalling pathway. This is the first report describing a regulatory role of HMGA1 on Akt signalling in a cancer model. Numerous reports have described HMGA1 overexpression to be prevalent in a wide range of human malignancies. Increasingly, tumoural HMGA1 expression status is reported to have prognostic value among patients with cancer (Sarhadi et al, 2006). Recently, data suggesting mechanisms by which HMGA1 may mediate cancer progression have begun to emerge. For example, HMGA1 proteins have been demonstrated to promote tumour progression and epithelial -mesenchymal transition in human breast epithelial cells (Reeves et al, 2001). In immortalised rat embryonic fibroblast Rat1a cells, HMGA1 was found to be a c-Jun-target gene and its suppression by antisense methodology was found to reduce the ability of c-Jun-overexpressing cells to grow under anchorage-independent conditions (Hommura et al, 2004).
Although previous reports have implicated HMGA1 as having the capacity to promote tumourigenesis and anchorage-independent growth in normal epithelial cells (Reeves et al, 2001), no studies reported to date have examined specifically the role of HMGA1 in mediating anoikis resistance in the context of cancer cells. Our findings suggest that HMGA1 overexpression represents a molecular determinant of anoikis resistance in pancreatic adenocarcinoma cells. The biological significance of this finding relates to emerging understanding that anoikis resistance is a phenotypic hallmark of metastatic cancer cells (Chuma et al, 2004;Douma et al, 2004). Although the underlying mechanisms rendering anoikis resistance in cancer cells are incompletely understood, our findings provide evidence for a novel role for HMGA1 in mediating this process. Further, our findings provide a potential mechanism by which HMGA1 overexpression induces anoikis resistance: activation of the antiapoptotic PI3-K/Akt signalling pathway.
Description of regulatory roles for HMGA1 in the context of apoptosis is not without precedent. HMGA1 has been found to associate in vivo with p53 family members and inactivate their functions. As such, overexpression of HMGA1 may lead to suppression of p53-induced apoptosis and tumour-suppressing activity (Frasca et al, 2006). In addition, antisense-mediated suppression of HMGA1 expression in thyroid carcinoma and pancreatic adenocarcinoma cell lines has been reported to induce an apoptotic response (Scala et al, 2000;Trapasso et al, 2004). Our findings that HMGA1 overexpression results in constitutive activation of PI3-K/Akt pathway provide another mechanism through which HMGA1 mediates its antiapoptotic functions. HMGA1-dependent activation of Akt signalling is likely to protect cancer cells from broad array of proapoptotic stimuli, not just those related to anchorage deprivation. Indeed, we have recently reported that HMGA1 protects pancreatic adenocarcinoma cells from gemcitibine-induced apoptosis and in this context is a potential molecular determinant of chemoresistance in pancreatic cancer (Liau et al, 2006).
Our findings, taken together with those previously reported, provide a mechanistic basis for conceptualising the known correlations between HMGA1 expression and poor prognosis in cancer patients (Chiappetta et al, 2004). These findings also have obvious therapeutic implications. Future studies will need to address whether targeted therapies directed against HMGA1 will inhibit metastasis in pancreatic cancer.