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High mobility group A1 protein expression reduces the sensitivity of colon and thyroid cancer cells to antineoplastic drugs
© D’Angelo et al.; licensee BioMed Central Ltd. 2014
Received: 5 May 2014
Accepted: 6 November 2014
Published: 20 November 2014
Development of resistance to conventional drugs and novel biological agents often impair long-term chemotherapy. HMGA gene overexpression is often associated with antineoplastic drug resistance and reduced survival. Inhibition of HMGA expression in thyroid cancer cells reduces levels of ATM protein, the main cellular sensor of DNA damage, and enhances cellular sensitivity to DNA-damaging agents. HMGA1 overexpression promotes chemoresistance to gemcitabine in pancreatic adenocarcinoma cells through an Akt-dependent mechanism.
To elucidate the role of HMGA1 proteins in chemoresistance we analyzed resistance to conventional drugs and targeted therapies of human colon carcinoma cells (GEO) that are sensitive to the epidermal growth factor receptor inhibitor cetuximab, and express minimal levels of HMGA1 and cetuximab-resistant (GEO CR) cells expressing high HMGA1 protein levels.
GEO CR cells were less sensitive than GEO cells to cetuximab and 5-fluorouracil. GEO CR cells silenced for HMGA1 expression were more susceptible than empty vector-transfected cells to the drugs’ cytotoxicity. Similar results were obtained with anaplastic thyroid carcinoma cells expressing or not HMGA1 proteins, treated with doxorubicin or the HDAC inhibitor LBH589. Finally, HMGA1 overexpression promoted the DNA-damage response and stimulated Akt phosphorylation and prosurvival signaling.
Our findings suggest that the blockage of HMGA1 expression is a promising approach to enhance cancer cell chemosensitivity, since it could increase the sensitivity of cancer cells to antineoplastic drugs by inhibiting the survival signal and DNA damage repair pathways.
Chemotherapy is one of the most effective tools for the treatment of neoplastic diseases, but it has two relevant drawbacks: 1) it can harm normal cells, and 2) relapse often occurs within 5 years, and recurrent disease is frequently much more resistant to chemotherapy. The advent of new drugs that selectively target specific molecular pathways involved in tumorigenesis or tumor progression, known as “targeted therapy”, has improved patient outcome and survival. However, both conventional chemotherapy and targeted therapies can fail because of acquired drug resistance. Several mechanisms alone or in combination can confer resistance to cancer cells, namely amplification of cell survival signal pathways, increased DNA damage repair, and altered cellular drug uptake, efflux or metabolism [1, 2]. However, each mechanism only partially justifies the lack of response observed in cancer patients. Thus, the identification of other mechanisms mediating drug resistance is a challenge of oncological research.
High Mobility Group A (HMGAs) proteins are small non-histone chromatin factors that bind the minor groove of AT-rich DNA sequences through three N-terminal basic domains called “AT-hooks”. The HMGA family consists of four members: HMGA1a, HMGA1b and HMGA1c (which are encoded through alternative splicing by the HMGA1 gene) and HMGA2 (encoded by the HMGA2 gene) [3, 4]. HMGAs are highly expressed during embryogenesis, and low or absent in normal adult tissues. They are overexpressed in almost all human malignant neoplasias, often associated with metastases and a poor prognosis . HMGA proteins play a key role in chemoresistance. Indeed, HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy . HMGA1 overexpression promotes chemoresistance to gemcitabine in pancreatic adenocarcinoma cells in vitro through an Akt-dependent mechanism. Moreover, HMGA1-silencing promotes gemcitabine-induced cytoxicity and reduces tumor growth in vivo in a nude mouse xenograft model of pancreatic cancer .
Our group also demonstrated the involvement of HMGAs in the pathway of Ataxia-Teleangiectasia-Mutated (ATM) protein, the main cellular sensor of DNA damage. We demonstrated that HMGA proteins positively regulate ATM expression and the inhibition of HMGA1 expression through an antisense approach drastically decreases cellular levels of ATM in anaplastic thyroid cancer (ATC) cells, resulting in increased sensitivity to genotoxic agents .
To determine the role of HMGA1 proteins in chemoresistance we have analyzed the resistance to antineoplastic drugs of (i) the human colon carcinoma cells (GEO) that are sensitive to the epidermal growth factor receptor (EGFR) inhibitors cetuximab (CTX) and gefitinib, and that express barely detectable levels of HMGA1, and (ii) CTX-resistant GEO (GEO CR) cells that express high HMGA1 protein levels and are generated through in vivo continuous treatment with the drug followed by tumor explant and in vitro stabilization of the deriving resistant cancer cell lines .
Drugs and treatment
Cetuximab was purchased from ImClone Systems.
Doxorubicin and 5-Fluorouracil were purchased from Sigma (Sigma Aldrich, St Louis, MO, USA).
LBH589 was kindly provided by Dr. Caraglia. For ATM inhibition experiments, cells were treated with KU-55933 (Calbiochem) (10 μM) for 1 h before the induction of ATM kinase activity.
Cell lines, expression vector and transfection
Human GEO and SW48 colon cancer cells and FRO thyroid anaplastic carcinoma cells were from the American Type Culture Collection (Manassas, VA, USA). GEO CR (CTX resistant) cells were established as described previously . Hairpin RNA interference plasmids were from The RNAi Consortium (Sigma Aldrich). The control PLKO.1 plasmid, which has a scrambled non-targeting short-hairpin (sh) RNA sequence, was from SIGMA. FRO shHMGA1, GEO CR shHMGA1 and respective sh NoTargeting control stable clones were generated by transfection of the above indicated plasmids using the Neon™ Transfection System (Life Technologies, Carlsbad, California). The pCEFLHA and the pCEFLHA-HMGA1, vectors are described elsewhere . GEO pCEFL-HA, GEO-HMGA1, SW48 pCEFL-HA and SW48-HMGA1 cells were generated by transfection of the above-indicated plasmids using the Neon™ Transfection System.
Cells were transfected using Neon™ Transfection System (Life Technologies, Carlsbad, California) under the following conditions:
FRO: Pulse voltage (v): 1450, Pulse Width (ms): 10, Pulse number: 3;
GEO and SW48: Pulse voltage (v): 1300, Pulse Width (ms): 30, Pulse number: 1.
After transfection, stable clones were selected by exposure to 1 μg/ml of puromycin (GEO CR and FRO) or 800 ng/ml of neomycin (GEO and SW48) in complete medium.
Protein extraction, western blotting and antibodies
Cells were lysed in lysis buffer containing 1% NP40, 1mM EDTA, 50mM Tris–HCl (pH 7.5) and 150mM NaCl, supplemented with complete protease inhibitors mixture (Roche, Branford, CT, USA). Total proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham, Piscataway, NJ, USA). Membranes were blocked with 5% non-fat dry milk and incubated with the following antibodies: anti-HMGA1 polyclonal antibody, as previously described , anti-ATM S1981p (Rockland, Philadelphia, PA, USA), anti-ATM (Ab91) (Abcam Cambridge, MA) β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), (SC-1615; Santa Cruz), anti-Akt, anti Akt S473p and Phospho-p70 S6 Kinase (Thr389) (Cell Signaling, Beverly, MA, USA), anti Caspase-3 (Santa Cruz), anti-phospho-H2AX (ser 139) (Upstate Biotechnology, Lake Placid, NY, USA).
RNA extraction and quantitative-RT-PCR
Total RNA was isolated using TRI-reagent solution (Sigma) and reverse transcription was performed according to standard procedures (Qiagen, Valencia, CA, USA). qRT-PCR analysis was performed using the following primers:
HMGA1 Fw: 5′-CAACTCCAGGAAGGAAACCA-3′;
HMGA1 Rv: 5′-AGGACTCCTGCGAGATGC-3;
β-actin Fw: 5′- CCAACCGCGAGAAGATGA-3;
β-actin Rv: 5′-CCAGAGGCGTACAGGGATAG -3.
Primers for β-actin were used to normalize qRT-PCR data. To calculate the relative expression levels we used the 2-ΔΔCT method .
Cell viability assay
Drug-induced cytotoxicity was quantified by MTS (3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega’s CellTiter® 96 AQueous One Solution, Promega Fitchburg, WI, USA). Cells were seeded in 96-well plates at 5×103 cells per well, then exposed to serial dilutions of the drugs. After 72h absorbance was measured at 490 nm.
Cells were treated with 5FU or doxorubicin and apoptosis was quantified by measuring Caspase 3/7 activation using the Caspase-Glo 3/7 assay (Promega).
Cells were treated with CTX and allowed to repair the DNA for 0, 18 and 24 hours and then processed for the COMET assay (Trevigen, Helgerman, CT, USA) following manufacturer’s instructions. Cell images were analyzed using COMET Score (TriTek, Annandale, VA, USA). Comet tail moment was used as the measure of DNA damage. In each experiment, 50 comets were measured per experimental point and the mean ± S.D. was reported.
We used two-way analysis of variance to compare inter-group differences. The significance of differences was determined by analysis of variances followed by Dunnett’s test as post hoc test using Graph Pad Prism 5.0. Data are reported as mean ± SD and p <0.05 was accepted as statistically significant.
HMGA1 overexpression correlates with increased resistance to drug-induced cytotoxicity
Moreover, we examined the sensitivity to these drugs of the colon carcinoma cell line, SW48, which expresses low HMGA1 levels, transfected with HMGA1 (SW48-HMGA1) (Additional file 1: Figure S1A). As shown in Additional file 1: Figure S1B, SW48-HMGA1 cells were more resistant to the effect of CTX and 5FU than the empty-vector-transfected SW48 cells (SW48 pCEFL-HA cells).
Overexpression of HMGA1 correlates with activation of the prosurvival pathway
Overexpression of HMGA1 correlates with activation of the DNA-damage response
It is known that EGFR stimulate DSB repair after irradiation or activation by its ligands  and that the ability of tumor cells to repair DNA damage is reduced following EGFR blockade with Cetuximab . Then, to test whether HMGA1 was able to affect DSB repair after CTX exposure, GEO CR cells expressing or not HMGA1 were treated with CTX, and a comet assay was performed to evaluate the DNA-repair ability following drug exposure. Cells were collected after 0, 18 and 24 hours of treatment and the amount of damaged DNA in each cell type was analyzed evaluating the comet tail moment as a measure of the DNA damage. Interestingly HMGA-silenced cells displayed higher levels of DNA damage after exposure to CTX for 18 hours, compared with GEO CR empty vector. Moreover, after 24 hours HMGA-overexpressing cells were able to almost completely repair the damage, while HMGA-silenced cells still showed significant levels of damaged DNA (Figure 6C).
These data suggest that DSB repair ability is affected by the presence of HMGA1 proteins also after CTX treatment.
Overexpression of HMGA1 and HMGA2 is a general feature of experimental and human malignancies and their overexpression is often correlated with aggressiveness, resistance to conventional anti-cancer therapies and poor prognosis .
In this study we have evaluated the role of HMGA1 proteins in resistance to both conventional and biological antineoplastic drugs. We found that enforced overexpression of HMGA1 in human colon carcinoma cells GEO, sensitive to CTX and expressing low levels of HMGA1, promoted resistance to CTX and 5FU. Conversely HMGA1-silencing on GEO CR cells abrogated resistance to the above indicated drugs. Similar findings were obtained with FRO cells expressing high HMGA1 levels treated with doxorubicin and LBH589. Accordingly, the HMGA1-silenced cells displayed a higher apoptotic rate and caspase 3/7 activation after exposure to 5FU or doxorubicin. This result is consistent with our previous data showing that the block of HMGA1 expression obtained by using an adenovirus carrying the HMGA1 cDNA in antisense orientation leads thyroid carcinoma cells to apoptotic death .
To determine the mechanisms by which HMGA1 promotes drug resistance, we analyzed the Akt-dependent prosurvival signaling. Indeed, previous findings demonstrate that HMGA1 overexpression mediates gemcitabine resistance in pancreatic adenocarcinoma cells through an Akt-dependent mechanism , and over-activation of Akt pathway is a poor prognostic factor in cancer . Here, consistent with previous results, we report that HMGA1 silencing inhibits activation of the Akt pathway after treatment with 5FU or doxorubicin.
We next investigated whether the reduced sensitivity of the HMGA1-overexpressing cells was associated with enhancement of the DNA-damage response, since we previously demonstrated that HMGA1 positively regulates cellular levels of ATM in anaplastic thyroid cancer cells so causing reduced sensitivity to genotoxic agents . Functional interactions have been identified between ATM and growth factor-mediated signaling . In fact, ATM is a nuclear protein kinase that functions as a signal transducer in response to DNA damage, but has also a cytoplasmic localization mediating the activation of Akt through a growth factor-mediated signaling pathway . Therefore, the decreased ATM levels in the presence of HMGA1 overexpression might increase pro-survival Akt signaling after treatment of the cancer cells with the antineoplastic drugs.
It has been reported that stimulation of EGFR after irradiation or activation by its ligands, such as EGF or TGFalpha activate DSB repair . The ability of tumor cells to repair DNA damage is reduced following EGFR blockade with Cetuximab , and recently it has been demonstrated that the combination of EGFR inhibition and DNA damage-induced therapy increases in vitro and in vivo response of human tumor cells .
Here, we report that the ability of GEO CR cells, resistant to CTX, to repair DNA damage is reduced in absence of HMGA1 proteins. These data support the idea that HMGA1, inducing an overactivation of DNA damage response, makes less effective the blockade of this pathway by CTX. This mechanism could act in combination with the overactivation of PI3K/AKT prosurvival pathway, widely reported upregulated in CTX-resistant cells .
In conclusion, our findings suggest that the block of HMGA1 proteins could increase the sensitivity of cancer cells to antineoplastic drugs by inhibiting the survival signal and DNA damage repair pathways, the overactivation of which is a hallmark of resistance to anticancer therapies and a poor prognostic factor in cancer progression. Therefore, the targeted suppression or inactivation of HMGA1 could be a potential therapeutic strategy with which to increase chemosensitivity in cancer cells.
This work was supported by grants from AIRC (IG 11477) and the Ministero dell’Università e della Ricerca Scientifica e Tecnologica–MIUR (PRIN2008). DD is recipient of a fellowship from the Fondazione Italiana per la Ricerca sul Cancro (FIRC). We thank Mario Berardone for the art-work. We are grateful to Jean Ann Gilder (Scientific Communication srl., Naples, Italy) for substantial editing of the text.
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