TMP269 | Gsk3 Signal

FOXO3a Activation by HDAC Class IIa Inhibition Induces Cell Cycle Arrest in Pancreatic Cancer Cells

Makoto Usami, MD,* Shohei Kikuchi, MD, PhD,*† Kohichi Takada, MD, PhD,* Michihiro Ono, MD, PhD,* Yusuke Sugama, MD,* Yohei Arihara, MD, PhD,* Naotaka Hayasaka, MD, PhD,*
Hajime Nakamura, MD, PhD,* Yuuki Ikeda, MD,* Masahiro Hirakawa, MD, PhD,* Makoto Yoshida, MD, PhD,* Koji Miyanishi, MD, PhD,*
Masayoshi Kobune, MD, PhD,† and Junji Kato, MD, PhD*

Objectives: Pancreatic cancer (PC) is highly aggressive with multiple on- cogenic mutations. The efficacy of current chemotherapy is poor, and new therapeutic targets are needed. The forkhead box (FOX) proteins are mul- tidirectional transcriptional factors strongly implicated in malignancies. Their expression is consistently suppressed by several oncogenic pathways such as PI3K/AKT signaling activated in PC. A recent study showed that class IIa his- tone deacetylases (HDAC) can act as a transcriptional suppressor. In this study, we hypothesized that HDAC class IIa inhibition would upregulate FOXO3a expression, thereby inducing its transcription-dependent antitumor effects. Methods: We confirmed the change of FOXO3a expression and the effect of the cell growth inhibition by HDAC class IIa inhibition in AsPC-1 cells. Be- cause FOXO3a is subject to ubiquitylation-mediated proteasome degradation, we examined the synergistic activation of FOXO3a by HDAC class IIa selec- tive inhibitor TMP269 combined with proteasome inhibitor carfilzomib.

Results: We observed that TMP269 induced FOXO3a expression in a
dose-dependent manner and inhibited cell growth in AsPC-1 cells. G1/S arrest was observed. FOXO3a expression was further increased and cell growth inhi- bition was dramatically enhanced by TMP269 combined with carfilzomib. Conclusions: Dual inhibition of class IIa HDACs and proteasome could be a promising new strategy for modifying FOXO3a activity against PC.
Key Words: FOXO3a, class IIa HDACs, cell cycle arrest, HDAC inhibitor, pancreatic cancer
(Pancreas 2020;49: 135–142)

Pancreatic cancer, which commonly manifests as a ductal ade- nocarcinoma, is a highly aggressive malignancy that is the fourth leading cause of cancer-related deaths in the United States and Japan.1,2 Surgical resection is the only potentially curative treat- ment option; however, most patients are diagnosed when the cancer
From the Departments of *Medical Oncology and †Hematology, Sapporo Med- ical University School of Medicine, Sapporo, Japan.
Received for publication June 9, 2019; accepted November 12, 2019. Address correspondence to: Junji Kato, MD, PhD, S-1, W-16, Chuo-Ku,
Sapporo 060-8543, Japan (e‐mail: [email protected]).
M.U. and S.K. contributed equally to this work.

This work was supported by JSPS KAKENHI Grant Number JP16K09399. The authors declare no conflicts of interest.
M.U., S.K., K.T., and M.O. Conception and design; M.U., S.K., and K.T. contributed to the development of methodology; M.U. and S.K., acquisition of data; M.U., S.K., K.T., M.O., Y.S., Y.A., N.H., H.N., Y.I., M.H., and
M.Y., analysis and interpretation of data; M.U. and S.K., writing, review, and/or revision of the manuscript; M.U. and S.K., administrative, technical, or material support; and K.M., M.K., and J.K., study supervision.

Supplemental digital contents are available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.pancreasjournal.com).
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/MPA.0000000000001462
is locally advanced or metastatic and unresectable. Although sys- temic cytotoxic chemotherapy is given, its therapeutic efficacy is poor. The addition of the molecular targeting agents such as erlo- tinib and bevacizumab, which have clinical benefit against other malignancies such as lung cancer and colorectal cancer,3,4 has had lit- tle effect in pancreatic cancer, despite the importance of their cellular targets during disease progression.5,6 Pancreatic adenocarcinoma is accompanied by multiple genetic mutations, including those in KRAS and p53, which are associated with disease progression and chemoresistance.7–9 The presence of multiple alterations is one of the main reasons that single molecular targeting agents do not improve clinical outcome. Thus, novel agents that can im- prove clinical outcome are urgently needed; ideally, the effects of such agents will be sufficiently broad as to overcome the onco- genic effects of multiple genetic mutations.

The forkhead box O (FOXO) proteins are a subgroup of the forkhead box (FOX) family and function as multidirectional tran- scriptional factors that control a wide spectrum of biological pro- cess,10,11 including cell growth, survival, metabolism, and oxidative state.12 Although FOXO proteins, and particularly FOXO3a, nor- mally act as tumor suppressors via their activation of cell cycle ar- rest and apoptosis,13,14 they can be constitutively inactivated by several major oncogenic signaling pathways, such as PI3K/ AKT, MAPK, Ras-MEK-ERK, and IKK pathways.13,15,16 In pancreatic cancer, the PI3K/AKT pathway is frequently activated,17 leading to inactivation of FOXO proteins, cancer cell survival, and chemoresistance. Thus, upregulating FOXO3a activity could be a promising strategy for pancreatic cancer treatment independently of the underlying oncogenic mutations. However, targeting tran- scription factors is a relatively novel approach and is challenging in terms of tractability.

Histone deacetylases (HDACs) can be divided into four groups (class I: HDAC1, 2, 3 and 8; class IIa: HDAC4, 5, 7 and 9; class IIb: 6 and 10; class III: SIRT 1–7; class IV: HDAC11).18 Class IIa HDACs are quite distinct, as they have nucleus-cytoplasmic shuttling ca- pability and minimal deacetylase activity when compared with nuclear class I HDCAs and cytoplasmic class IIb HDACs.19 His- tone deacetylases inhibitors (HDACi) have been extensively in- vestigated and used clinically for cancer treatment, and their major mechanism of action is considered to be the modulation of histone modification, predominantly as a result of their activity against class I HDACs. The biological significance of class IIa HDACs in cancer is not fully understood. Recent studies have in- dicated that class IIa HDACs repress the transcription factors. In a preclinical study, the selective HDAC class IIa inhibitor, TMP269, induced upregulated transcription factor ATF4 followed by induc- tion of apoptosis in multiple myeloma cells.20 The expression and activity of FOXO proteins are mainly negatively regulated through their AKT-dependent phosphorylation, which facilitates their binding to 14-3-3 proteins and their subsequent export to the cytoplasm.12,21

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Indeed, 14-3-3 proteins are pivotal interacting partners of class IIa HDACs.19,22 Thus, targeting class IIa HDACs has great potential as a strategy to restore the activity of FOXO proteins.
In this study, we investigated the expression of FOXO3a in pan- creatic cancer cells and tested our hypothesis that class IIa HDACs in- hibition would upregulate FOXO3a expression and induce antitumor effects by restoring FOXO3a transcriptional activity. Our results provide the rationale for HDAC class IIa inhibitor–based therapeutic strategies designed to restore FOXO3a activity in pancreatic cancer.

MATERIALS AND METHODS
Cell Lines and Culture Conditions
The pancreatic cancer cell lines AsPC-1 and BxPC-3 were purchased from American Type Culture Collection (Manassas, Va). MIA PaCa-2 and PANC-1 were obtained from the Riken BRC Cell Bank (Tsukuba, Japan). All cell lines were cultured with RPMI 1640 medium (Sigma-Aldrich, St Louis, Mo) containing 10% fetal bovine serum (GIBCO, Thermo Fisher Scientific, Waltham, Mass), 2 μM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich).

Reagents
The selective HDAC class IIa inhibitor TMP269 was purchased from Selleck Chemicals (Houston, Tex) and the proteasome inhibitor carfilzomib (CFZ) from LC Laboratories (Woburn, Mass).

Immunoblotting
Cells were harvested, washed with phosphate-buffered saline, and lysed using RIPA buffer containing protease inhibitor cocktail (Roche, Indianapolis, Ind). Whole cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trans- ferred to polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany), and immunoblotted with the following an- tibodies: anti-HDAC4, HDAC7, FOXO3a, PARP, caspase-3, CDK2, CDK4, CDK6, Cyclin E1, Cyclin E2, Cyclin D1, and Cyclin D3 (Cell Signaling Technology, Danvers, Mass), and anti-HDAC5, HDAC9, p21, and actin (Santa Cruz Biotechnology, Dallas, Tex). Protein expression level was evaluated by standard chemilumines- cence using ImageQuant LAS-4000 (GE Healthcare, Chicago, Ill) and quantified by ImageJ version 1.50i software (NIH, Bethesda, Md).

Quantitative Reverse Transcription-Polymerase Chain Reaction and Polymerase Chain Reaction Arrays
AsPC-1 cells were cultured for 48 hours with the designated dose of TMP269, or in control complete media, and harvested. Total RNA was purified with the RNeasy Plus Mini Kit (QIAGEN, Ger- mantown, Calif ) according to the manufacturer’s instructions. Re- verse transcription was carried out using SuperScript VILO Master MIX (Thermo Fisher Scientific). Quantitative reverse transcription– polymerase chain reaction (PCR) was performed with an Applied Biosystems 7300 Real-time PCR system (Applied Biosystems, Foster City, Calif ). Analysis of target genes was conducted in trip- licate using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Transcript levels were normalized to β-actin expression. The PCR primers were designed: 5′-CGGACAAACG GCTCACTCT – 3′ and 5′-GGACCCGCATGAATCGACTAT-3′ for FOXO3; 5′-GGCATCCTCACCCTGAAGTA-3′ and 5′-
GAAGGTGTGGTGCCAGATTT-3′ for β-actin. For PCR array, total RNA was reverse transcribed using RT2 First Strand Kit (QIAGEN). Polymerase chain reaction array was performed using RT2 Profiler PCR Array Human PI3K-AKT Signaling Pathway according to the manufacturer’s instruction.

Growth Inhibition Assay
The growth inhibitory effect of TMP269, alone or with CFZ, was assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide (MTT; Sigma-Aldrich) dye absorbance. Briefly, 5000 AsPC-1 cells were cultured with 100 μL of complete medium in each well of 96-well culture plate. Twenty-four hours later, supernatants were removed and 100 μL of complete media containing the designated dose of TMP269 and/or CFZ was added into each well. Ten microliters of 5 mg/mL MTTwas added to each well for the last 4 hours of the 48-hour treatment. Ab- sorbance was measured at 570 nm with absorbance at 650 nm subtracted as a reference using a multimode plate reader (Infinity M1000 PRO; TECAN, Männedorf, Switzerland). All experiments were performed in triplicate.

Cell Cycle Assay
ImageImageImageImageImageImageImageImageImageImageImageImageImageAsPC-1 cells cultured with or without TMP269 for 48 hours were harvested, washed with phosphate-buffered saline, and fixed FOXO3a expression in pancreatic cancer. Publicly available gene expression profiling data GSE 62125 (A) and GSE28735 (B) were analyzed. A, FOXO3a expression was significantly lower in 118 resected pancreatic ductal adenocarcinoma (PDAC) tissue samples than in 13 normal pancreatic tissues (P < 0.0001)B, FOXO3a expression was significantly lower in cancer tissue compared with adjacent noncancer normal tissue of resected pancreas in 45 pancreatic ductal adenocarcinoma patients (P = 0.0015). Differences in overall survival among the low expression group (FOXO3a level under the 25th percentile of 63 patients, redline), middle expression group (25th–75th percentile, black line), and high expression level (above the 75th percentile, blue line). C,Median survival times of the low-, middle-, and high-expression groups were 13, 21.5, and 33 months, respectively 136 www.pancreasjournal.com © 2019 Wolters Kluwer Health, Inc. All rights reservedwith 70% of ethanol. Cells then were stained with 50 μg/mL propidium iodine solution (Sigma-Aldrich). After flow cytometry on a FACS Canto II instrument, data were analyzed using BD FACS Diva software (BD Biosciences, San Jose, Calif ). All ex- periments were performed in triplicate.

Statistical Analysis
Statistical significance was determined using the Student t test.
P < 0.05 was considered to indicate statistical significance.

RESULTS
FOXO3a Expression Is Suppressed in Pancreatic Cancer
We first investigated FOXO3a expression in pancreatic can- cer patients using publicly available gene expression profiling
data. FOXO3a expression was significantly lower in 118 resected pancreatic adenocarcinoma tumor tissues than in 13 normal pan- creatic tissues (Fig. 1A).23 Furthermore, in paired analysis be- tween cancer tissue and noncancer tissue in the same pancreatic ductal adenocarcinoma patient, FOXO3a expression was signifi- cantly lower in the cancer tissues (Fig. 1B).24 To investigate the impact of FOXO3a expression on survival, we analyzed gene ex- pression profiling data from a retrospective microarray study of 63 resected early-stage pancreatic adenocarcinoma patients.25 We divided the 63 patients into 3 groups according to FOXO3a expression level (low expression group: FOXO3a expression under the 25th percen- tile, middle expression group: 25th–75th percentile, high expression group: above the 75th percentile; Supplemental Fig. 1, http://links. lww.com/MPA/A760). The median survival of the low expression group was 13 months less than that of the middle expression group (21.5 months) and high expression group (33 months;

HDAC class IIa inhibition induces FOXO3a upregulation. Cell lysates were obtained from AsPC-1, MIA PaCa-2, Panc-1, and BxPC-3 pancreatic cancer cell lines. Cell lysates were immunoblotted with indicated antibodies. A, β-Actin was used as a loading control. B, Gene expression levels of HDAC4, HDAC5, HDAC7, and HDAC9 as obtained from the publicly available gene expression profiling data set, GSE 15471. Heatmap displaying the differentially expressed genes of the PI3K-AKT pathway in the AsPC-1 pancreatic cancer cell line. Data of the indicated genes were collected from the PCR array performed with AsPC-1 in the presence or absence of TMP269. C, FOXO3 (position: C02) was the fifth most upregulated gene among 84 examined genes, with a 1.99-fold increase. D and E, FOXO3a messenger RNA and protein expression was increased in a dose-dependent fashion.

TABLE 1. Upregulated Genes Triggered by HDAC Class IIa Inhibition in AsPC-1

Position RefSeq No. Gene Symbol Description Fold Regulation
B10 NM_000639 FASLG Fas ligand (TNF superfamily, member 6) 3.31
C04 NM_005311 GRB10 Growth factor receptor-bound protein 10 2.65
E03 NM_002576 PAK1 P21 protein (Cdc42/Rac)-activated kinase 1 2.43
G10 NM_000548 TSC2 Tuberous sclerosis 2 2.01
C02 NM_001455 FOXO3 Forkhead box O3 1.99
D07 NM_002746 MAPK3 Mitogen-activated protein kinase 3 1.97

indicating that low FOXO3a expression is an indicator of poor survival. These findings indicate that FOXO3a upregulation might be a therapeutic strategy for pancreatic cancer treatment.

HDAC Class IIa Inhibition Induces FOXO3a Upregulation
ImageImageImageImageImageImageImageImageBecause class IIa HDACs have tissue-specific expression,19 we investigated their expression in pancreatic cancer cell lines (Fig. 2A), and in publicly available gene expression profiling data of pancreatic adenocarcinoma cancer tissue and noncancer tissue (Fig. 2B).26 Expression of all isozymes was confirmed in the celllines we tested (Fig. 2A), and in pancreatic cancer and noncancer tissue (Fig. 2B). We next used a potent cell permeable and selective class IIa HDAC inhibitor (TMP269; C25H21F3N4O3S)27 to probe the role of this HDAC subfamily in pancreatic cancer. In the PCR ar- ray assay, FOXO3 was the fifth most upregulated gene among 84 tested (1.99-fold increase, Fig. 2C and Table 1). Importantly, FASLG, a FOXO3a target, was the most upregulated gene, strongly suggest- ing that FOXO3a transcriptional activity was increased upon treat- ment. We confirmed that FOXO3a expression at the messenger RNA and protein level was increased in a dose-dependent manner after treatment with TMP269 (Figs. 2D, E). These findings confirm that HDAC class IIa inhibition induces FOXO3a upregulation.

TMP269 induces cell growth inhibition effect. A, AsPC-1, MIA PaCa-2, BxPC-3, and Panc-1 were treated with the indicated doses of TMP269 (0–100 μM) for 48 hours, and cell growth was assessed using the MTT assay. Data represent mean (standard deviation) in triplicate. B, AsPC-1 cells were treated with TMP269 (0–50 μM) for 48 hours. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as the loading control. C, Cell cycle distribution was analyzed using flow cytometric analysis.Image138 www.pancreasjournal.com © 2019 Wolters Kluwer Health, Inc. All rights reserved.

TMP269 Treatment Induces Modest Cell Growth Inhibition Effect Via Cell Cycle Arrest
To investigate the antitumor effect of HDAC class IIa inhibi- tion by TMP269, we performed an MTT-based cell growth assay. TMP269 treatment induced modest cell growth inhibition in AsPC-1 pancreatic cancer cells, with an IC50 of 57.5 μM; the IC50 was not achieved in other cell lines, even with the maximal dose of 100 μM for 48 hours (Fig. 3A). To elucidate the mecha- nism of cell growth inhibition, we evaluated induction of apopto- sis with immunoblotting and examined changes in the cell cycle using flow cytometry. Because the PCR array assay revealed up- regulated expression of FASLG, the gene encoding FAS ligand (Fig. 2C, Table 1, and Supplemental Table 1, http://links.lww. com/MPA/A760), we suspected that the apoptosis pathway may have been engaged. However, neither cleaved PARP nor caspase-3 was observed after immunoblotting (Fig. 3B). A cell cycle assay revealed that G1/S arrest was induced in a dose-dependent fashion (Fig. 3C), showing that cell growth inhibition was due to cell cycle arrest rather than apoptosis. On further examination of the relationship between FOXO3a and proteins related to G1/S cell cycle transition, we observed downregulation of CDK2, CDK4, and CDK6, cyclin D1, cyclin D2, and upregulation of p21Waf1/Cip1, consistent with induction of G1/S arrest and transcriptional activation of FOXO3a (Figs. 4A–C). Importantly, p21Waf1/Cip1 upregulation was observed in the AsPC-1 p53-null cell line, highlighting p53-independent upregulation. These findings suggest that upregulation and transcriptional activation of FOXO3a after treatment with HDAC class IIa inhibitors lead to inhibition of cell growth.

Combined Proteasome and HDAC Class IIa Inhibition Enhances FOXO3a Activation and Cell Growth Inhibition
Because FOXO3a is subject to ubiquitylation-mediated pro- teasome degradation, we examined the effects of the irreversible
proteasome inhibitor CFZ when combined with TMP269. We hy- pothesized that CFZ-dependent stabilization of FOXO3a would synergize with TMP269-dependent activation of FOXO3a-dependent transcriptional activity. As expected, FOXO3a expression was fur- ther increased when TMP269 was combined with CFZ (Fig. 5A). Importantly, treatment with CFZ alone did not increase FOXO3a expression, indicating that dual inhibition of HDAC class IIa and the proteasome was required to enhance FOXO3a activity. Con- comitant with the increased FOXO3a activity with dual inhibitor treatment, p21Waf1/Cip1 expression was further upregulated (Fig. 5B) and cell growth inhibition was dramatically enhanced (Fig. 5C). These data suggest that dual inhibition of HDAC class IIa and the proteasome is a promising strategy for achieving antiproliferative effects in pancreatic cancer.

DISCUSSION
In this study, we showed that cell cycle arrest is triggered by FOXO3a upregulation in pancreatic cancer cell lines. FOXO3a is a negative regulator of cell proliferation repressing the activity of cyclin D1 and D2 and inducing specific cell cycle inhibitors such as p21Waf1/Cip1.10,28 Consistent with earlier reports, we showed that cyclins were downregulated, whereas p21 was upregulated; these findings are compatible with transcriptionally activated FOXO3a. To date, the biological impact of FOXO3a expression on pancreatic cancer cell survival and/or chemoresistance has re- mained unclear. Here, we demonstrated that FOXO3a is downreg- ulated in pancreatic cancer tissues, and that there was a trend of poor survival in patients with low FOXO3a expression in tumors. FOXO3a expression is suppressed by several oncogenic pathways including PI3K/AKT signaling, which is constitutively activated in pancreatic cancer; FOXO3a expression is also lower in pancre- atic cancer cell lines than in the normal human immortalized pancreatic ductal epithelial cell line, HPDE6.29 In bladder cancer, high expression of FOXO proteins, including FOXO3, is report- edly correlated with better prognosis.30 Together with our results, TMP269 inhibits cell growth. AsPC-1 cells were treated with TMP269 (0–50 μM) for 48 h. Cell lysates were immunoblotted with the indicated antibodies. β-actin was used as loading control (A, B, and C).

TMP269 combined with CFZ enhances FOXO3a upregulation. AsPC-1 cells were treated with TMP269, CFZ, or both for 48 hours. Cell lysates were immunoblotted with FOXO3a (A) and p21 (B). β-Actin was used as loading control. C, Cell growth was assessed using the MTT assay. Data represent mean (standard deviation) in triplicatethese findings strongly suggest that loss of FOXO3a activity is impli- cated in the progression and survival of pancreatic cancer. It follows that FOXO3a upregulation may provide promising opportunities for developing effective treatments to target pancreatic cancer.

Class IIa HDACs are considered to function as repressors of transcription factors.31 They shuttle between cytoplasm and nucleus and, because of minimal enzymatic activity, act by interacting with other proteins and/or by recruiting other enzymatically active HDACs such as HDAC3.32 These features seem to be essential to the function of Class IIa HDACs as transcriptional co-repressors. Prior examples include the regulation of FOX proteins by HDAC7 and HDAC9.33 In cancer cells, class IIa HDACs are known to interact with other tran- scription factors. For example, HDAC4 interacts with PLZF-RARα in acute promyelocytic leukemia cells,34 and HDAC4 interacts with HIF-1α in renal carcinoma cells.35 To upregulate FOXO3a expression, we used TMP269, the first selective class IIa HDACi with a new trifluoromethyloxazodale moiety and high selectivity without class I and IIb activity.27 As expected, we observed FOXO3a upregulation upon HDAC class IIa inhibition using TMP269; however, the cell growth inhibitory effects of TMP269 were mod- est. We inferred that this was because upregulation of FOXO3a was in itself insufficient. Because the activity of FOX proteins is regulated by also posttranslational modifications such as ubiquitylation,11 we hypothesized that a proteasome inhibitor such as CFZ would synergize with TMP269 with respect to upregulation FOXO3a levels and activity.

In pancreatic cancer treatment, the clinical outcome of sys- temic chemotherapy is poor, with 39% of subjects achieving anobjective response rate and 11.1 months of median overall survival; this is true even with the intensive triplet regimen of fluorouracil plus oxaliplatin and irinotecan (FORFIRINOX).36 To improve clin- ical outcome in pancreatic cancer, molecular targeted therapies that have demonstrated efficacy compared with chemotherapy alone in other malignancies such as lung cancer or colorectal cancer3,4 have been tried, but all have fallen short of expectation. For example, erlotinib, a small-molecule tyrosine kinase inhibitor targeting the epidermal growth factor receptor that is often expressed in pancre- atic cancer,37 was tried in a combination therapy with gemcitabine in a phase III clinical trial. Although survival was improved com- pared with gemcitabine monotherapy, the increase in life expec- tancy was only 2 weeks (median survival, 6.2 versus 5.9 months; P = 0.038).5 Bevacizumab, which targets vascular endothelial growth factor, is also often expressed in pancreatic cancer.38 How- ever, it conferred no additional survival benefit when combined with gemcitabine in a phase III trial.6 These disappointing results are partly due to the fact that pancreatic adenocarcinoma is ac- companied by multiple genetic mutations such as KRAS and P53; these mutations are associated with more rapid tumor pro- gression and chemoresistance,7–9 and single molecular targeted agents are not likely to have an effect in such circumstances. From this point of view, modifying transcription factor activity down- stream of oncogenic mutations may offer a novel and promising strategy for reversing the tumorigenic state. Indeed, we showed that p21 was upregulated concomitant with induction of cell cycle arrest after FOXO3a upregulation in p53-null AsPC-1 cells. This shows that some of the tumor suppressor functions of p53 (such as p21-dependent cell cycle arrest) can be recovered, even when p53 itself has been functionally inactivated.
In conclusion, we show here that HDAC class IIa inhibition induced FOXO3a upregulation, resulting in cell cycle arrest in a pancreatic cancer cell line. These studies provide the framework for dual inhibition of the proteasome and class IIa HDACs as a novel treatment strategy for activation of the FOXO3a transcrip- tion factor in pancreatic cancer.

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