Lung cancer accounts for approximately 2 million new cases and 1.76 million deaths annually worldwide; thus, it belongs to the group of cancers with high incidence and mortality [1]. Non-small cell lung cancer (NSCLC), which constitutes approximately 85% of lung cancer cases, presents with metastasis in approximately 70% of cases, and metastasis is associated with death in more than 90% of cases [2]. Despite several studies on NSCLC treatment, the five-year survival rate remains low at 9% [3]. Thus, it is imperative to develop effective therapeutic strategies to enhance the diagnosis and treatment of NSCLC.

Epigenetic regulation involves chromosomal modification-based changes in gene expression without alterations to the DNA sequence itself [4]. This regulation plays a pivotal role in the development and progression of cancer. In particular, an aberrant epigenetic regulation observed in cancer cells is closely related to the initiation, promotion, and progression of drug-resistant malignant tumors [5]. Histone deacetylases (HDACs) are key enzymes that deacetylate histone proteins to regulate gene expression as a form of epigenetic modification. In total, 18 different types of HDACs have been identified, which not only affect histone proteins, but also considerably affect the gene expression and signaling pathways of various non-histone proteins [6]. In particular, HDAC6, which is classified under class IIb HDACs, is overexpressed in various cancers, including lung and breast cancer [7,8]. HDAC6 is thought to promote malignancy via deacetylation of several non-histone proteins, including α-tubulin, cortactin, and heat shock protein 90 (HSP90) [9]. The downregulation of HDAC6, which is overexpressed in cancer, shows promising antitumor effects [8,10-13]. Consequently, various HDAC inhibitors that target HDAC6 are being developed, with ACY-1215 and ACY-241 currently in clinical phases [9]. Notably, ACY-241 has been reported to exhibit higher selectivity than that of ACY-1215, a first-generation HDAC6 selective inhibitor [11], and it is being investigated as a potential therapeutic agent to effectively inhibit cancer metastasis and malignancy.

Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor activated under hypoxic conditions, and its expression is increased in various solid tumors. HIF-1α modulates gene transcription by binding to the hypoxia-response element (HRE) located in the promoter region of several genes [14]. In particular, HIF-1α affects the expression of genes involved in cancer malignancy-related processes, including cell division, angiogenesis, energy metabolism, and epithelial–mesenchymal transition (EMT) [15]. Increased levels of HIF-1α, particularly in lung cancer, promote EMT and consequently affect cancer progression and metastasis [16]. Stabilized HIF-1α under hypoxic conditions is involved in the EMT process via regulation of expression of key EMT markers, including Snail, Slug, Twist, and zinc finger E-box-binding homeobox 1 (ZEB1) [17]. Therefore, the regulation of HIF-1α activity may suppress lung cancer metastasis and improve therapeutic outcomes.

HIF-1α activity is generally affected by proteasomal degradation under normoxic conditions. However, HIF-1α stability can be maintained even in normoxia [18,19]. Previous studies have reported that HDAC6 has the potential to regulate the stability and transcriptional activity of HIF-1α under hypoxic conditions [20]. However, the association between HIF-1α and HDAC6 under normoxic conditions remains unclear. Therefore, the aim of this study was to determine the relationship between HDAC6 and HIF-1α under normoxic conditions and to investigate the phenomena that occur upon HDAC6 inhibition. We observed an increased expression of HIF-1α in cancer cell lines with overexpressed HDAC6 under normoxic conditions. Thus, we hypothesized that HDAC6 is involved in maintaining HIF-1α stability in normoxia.

Antibodies and reagents

ACY-241 was purchased from Cayman Chemical Company, dimethyl sulfoxide and MG-132 were obtained from Sigma‐Aldrich. Antibodies against HDAC6, acetylated α-tubulin (Ac-α-tubulin), α-tubulin, ZEB1, E-cadherin, and secondary antibodies were purchased from Cell Signaling Technology. An antibody against glyceraldehyde 3-phosphate (GAPDH) was purchased from Santa Cruz Biotechnology, while an antibody against HIF-1α was obtained from BD Biosciences.

Cell culture

The human breast cancer cell lines MCF-7 and SK-BR-3 as well as human lung cancer cell lines A549, H1650, H1975, and HCC827 were purchased from the American Type Culture Collection. MDA-MB-231 cells were acquired from the Korean Cell Line Bank. The breast cancer cell lines were cultured in RPMI-1640 medium supplemented with HEPES and L-glutamine (HyClone Laboratories), while the lung cancer cell lines were cultured in RPMI-1640 medium supplemented with L-glutamine (HyClone Laboratories), 10% fetal bovine serum (FBS) (Atlas Biologicals), and 1% penicillin–streptomycin (HyClone Laboratories). All cultures were maintained in a humidified incubator at 37°C with 5% CO₂.

Cell viability assay

Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay. MDA-MB-231 and HCC827 cells were plated at a density of 2 × 10³ cells per well in a 96-well plate, while H1975 cells were seeded at a density of 1 × 10³ cells per well in a 96-well plate. After overnight incubation, the cells were treated with ACY-241 or dimethyl sulphoxide and incubated for 48 h. Subsequently, the cells were incubated with 10 μl CCK-8 for 2 h. The absorbance was measured at 450 nm for CCK-8 and 650 nm for the reference using the FLUOstar Omega microplate reader (BMG Labtech).

Wound healing assay

H1975 cells were plated into six-well plates (4 × 10⁵ cells/well) and incubated overnight. When the cells showed approximately 80% confluence, a monolayer of cells was scratched using a pipette tip and washed with culture medium to remove debris. The cells were cultured in RPMI-1640 medium containing 2% FBS for 36 h. Images of the cell monolayers were acquired at 0, 24, and 36 h using a microscope (Olympus IX71, Olympus) with consistent settings.

Transwell migration and invasion assays

Cell invasion and migration assays were performed using a Transwell insert (8 μm PET membrane; Corning Life Sciences). For the migration assay, 4 × 10⁴ cells in medium containing 2% FBS along with ACY-241 or dimethyl sulphoxide were introduced into the upper wells, while medium containing 2% FBS and ACY-241 or dimethyl sulphoxide was inserted into the lower wells. In the invasion assay, Corning Matrigel Matrix was diluted in serum-free medium to a final concentration of 100 μg/ml, and 50 μl of the diluted Matrigel matrix was carefully added to the center of each Transwell. Following gel formation of the Matrigel matrix, cell seeding was performed using the same method as in the migration assay. After 36 h, non-invading cells were removed. The invading cells present in the lower surface of the membrane were fixed with methanol and stained with 0.1% crystal violet. Five different sections of the membrane were photographed using a microscope, and the cell counts were determined in these five areas to calculate the average.

Small interfering RNA (siRNA) transfection

Double-stranded siRNAs against human HDAC6 (NM_ 001321225.1) and HIF-1α (NM_001243084.1) were synthesized by Bioneer. Negative control siRNA was obtained from Bioneer (Cat. No. SN-1003). The specific siRNA sequences used in this study are as follows: HDAC6 forward: 5ʹ-CCGGUUUGCUGAAAAGGAA-3ʹ and reverse: 5ʹ-UUCCUUUUCAGCAAACCGG-3ʹ; and HIF-1α forward: 5ʹ-GUGGUUGGAUCUAACACUA-3ʹ and reverse: 5ʹ-UAGUGUUAGAUCCAACCAC-3ʹ. MDA-MB-231 and HCC827 cells were plated at a density of 5 × 10⁵ cells per well, whereas H1975 cells were seeded at a density of 2 × 10⁵ cells per well in 100-mm petri dishes. After overnight incubation, the cells were transfected with siRNA or negative control siRNA using the Oligofectamine transfection reagent (Invitrogen) according to the manufacturer’s protocol. The transfection efficiency of siRNA was determined via Western blotting.

Western blotting

MDA-MB-231 and HCC827 cells were plated at a density of 1 × 10⁶ cells per well, whereas H1975 cells were seeded at a density of 4 × 10⁵ cells per well in 100-mm petri dishes. Following overnight incubation, the cells were treated with ACY-241 for 48 h. Subsequently, the cells were lysed using a cold whole-cell lysis buffer supplemented with the phosphatase inhibitor cocktail Halt Protease (Thermo Fisher Scientific) and phenylmethylsulfonyl fluoride (Thermo Fisher Scientific). The proteins were loaded onto sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene fluoride membranes (GE Healthcare). Membranes were blocked with 2.5% skim milk in Tris-buffered saline containing Tween 20 detergent, and then incubated overnight at 4°C with primary antibodies. Membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h, visualized using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific), and then developed using LAS-3000 (Fujifilm) according to the manufacturer's instructions.

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIZOL reagent (Invitrogen). Subsequently, cDNA synthesis was performed in a reaction mixture consisting of M-MLV Reverse Transcriptase, RNase inhibitor, dNTPs, and Recombinant RNase Inhibitor (all from Promega) at 42°C for 1 h. The cDNA samples were assayed in duplicate for qRT-PCR using the TB Green Premix Ex Taq II kit (Tli RNaseH Plus) (Takara Bio), according to the manufacturer’s instructions. All reactions were conducted in triplicate, and the expression levels were normalized to that of the 18s rRNA gene. The amplification process involved a reaction cycle comprising 95°C for 30 sec, followed by 95°C for 5 sec and 60°C for 30 sec. The fluorescence signal was detected at the end of the cycle using the LightCycler480 II instrument (Roche). mRNA expression levels were normalized to that of 18s rRNA using the 2^–ΔΔCT method [21]. The primer sequences used are: HIF-1α forward: 5ʹ- ACCACCTATGACCTGCTTGG-3ʹ and reverse: 5ʹ-CATATCCAGGCTGTGTCGAC-3ʹ; ZEB1 forward: 5ʹ-TTCACAGTGGAGAGAAGCCA-3ʹ and reverse: 5ʹ-GCCTGGTGATGCTGAAAGAG-3ʹ; 18s rRNA forward: 5ʹ-TAGTCGCCGTGCCTACCA-3ʹ and reverse: 5ʹ-TGCTGCCTTCCTTGGATGT-3ʹ.

Statistical analysis

Statistical analysis was performed using the Student's t-test (two-tailed) or one-way ANOVA using GraphPad Prism 8 (GraphPad Software). The data are presented as the mean ± standard deviation from a minimum of three independent experiments. p < 0.01 was considered statistically significant for this study.

Knockdown of HDAC6 under normoxia reduced HIF-1α level

HIF-1α is a key transcription factor that is activated under hypoxia and plays a role in cancer progression; however, its expression can be detected even under normoxia [18,22]. Since HDAC6 can affect HIF-1α stability under hypoxia, we investigated the association between HDAC6 and HIF-1α under normoxia. Under normoxia, we found that three cancer cell lines that exhibited relatively higher HDAC6 expression (MDA-MB-231, H1975, and HCC827) also showed increased HIF-1α levels (Fig. 1A). To explore the relationship between increased HDAC6 and HIF-1α expression under normoxia, we performed a knockdown of HDAC6 and measured the HIF-1α expression level. Consequently, all three cell lines showed a reduction in HIF-1α level (Fig. 1B). We performed qRT-PCR to determine whether the decrease in HIF-1α expression associated with HDAC6 knockdown was at the mRNA or protein level. The results showed no change in HIF-1α mRNA level (Fig. 1C), confirming that HDAC6 knockdown led to a reduction in HIF-1α at the protein level. Therefore, our results show that HDAC6 knockdown under normoxia induces a decrease in HIF-1α protein level in MDA-MB-231, H1975, and HCC827 cell lines.

Figure 1. HDAC6 knockdown reduces the protein levels of HIF-1α under normoxic conditions.
(A) Western blot analysis of HDAC6 and HIF-1α levels under normoxic conditions in breast cancer and lung cancer cells. (B) Cells were transfected with siHDAC6 for 48 h. HDAC6, HIF-1α, Ac-α-tubulin, and α-tubulin expression levels were measured using western blot analysis. GAPDH was used as the loading control. (C) Cells were transfected with siHDAC6 for 48 h. HIF-1α expression was measured using qRT-PCR. The expression value was normalized to the 18s rRNA level. Statistical analysis was performed using Student’s t-test. Error bars present the mean ± standard deviation (n = 3). NS > 0.05 vs. siCON. HDAC6, histone deacetylase 6; HIF-1α, hypoxia-inducible factor 1 alpha; siHDAC6, small interfering RNA against HDAC6; Ac-α-tubulin, acetylated alpha tubulin; GAPDH, glyceraldehyde 3-phosphate; siCON, negative control siRNA; NS, non-significant.

ACY-241 treatment reduced HIF-1α protein level in H1975 cells

As HDAC6 knockdown induced a decrease in HIF-1α level, we verified this effect based on the inhibition of HDAC6 activity via treatment with ACY-241, a selective HDAC6 inhibitor [12]. In all three cell lines tested, the ACY-241-mediated inhibition of HDAC6 activity led to a dose-dependent reduction in cell viability (Fig. 2A). ACY-241 treatment also decreased HIF-1α level in all three cell lines, particularly in H1975 cells, which showed a dose-dependent decrease (Fig. 2B). To investigate whether HIF-1α undergoes proteasomal degradation by ACY-241, we treated it with ACY-241 followed by co-treatment with MG-132 to inhibit proteasomal degradation. It was observed that the reduction of HIF-1α by ACY-241 was reversed upon treatment with MG-132 (Fig. 2C). Thus, we confirmed that ACY-241 treatment decreased cell viability in the three cell lines via the inhibition of HDAC6 activity and concurrently reduced HIF-1α protein level.

Figure 2. ACY-241 decreases the HIF-1α protein level.
(A) After treatment with ACY-241 for 48 h, the cell viability was measured using the CCK-8 assay. Statistical analysis was performed using one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 6). ***p < 0.001 vs. CON. (B) Cells were treated with ACY-241 for 48 h, after which the protein levels of HDAC6, HIF-1α, Ac-α-tubulin, and α-tubulin were measured via western blot analysis. GAPDH was used as the loading control. (C) Cells were treated with ACY-241 for 48 h and with MG-132 4 h before sampling, after which the protein levels of HIF-1α were measured via Western blot analysis. GAPDH was used as the loading control. HDAC6, histone deacetylase 6; HIF-1α, hypoxia-inducible factor 1 alpha; Ac-α-tubulin, acetylated alpha tubulin; GAPDH, glyceraldehyde 3-phosphate; CON, control.

Reduction of HIF-1α level suppressed EMT in H1975 cells

HIF-1α plays a crucial role in cancer and affects cell proliferation, migration, and angiogenesis, all of which contribute to a poor prognosis [23]. Therefore, we investigated the mechanisms of action via which the reduction of HIF-1α level mediated by HDAC6 inhibition affects cancer cells. Since the decrease in HIF-1α level mediated by HDAC6 inhibition was the greatest in H1975 cells, subsequent experiments were conducted using the H1975 cell line. Our data revealed that HIF-1α knockdown suppressed cell proliferation in the H1975 cells (Fig. 3A). Additionally, in cell migration and invasion assays using a Transwell, the HIF-1α knockdown significantly reduced both cell migration and invasion (Fig. 3B). To determine the effect of HIF-1α knockdown on EMT-related markers, we examined the protein expression of E-cadherin and ZEB1. Western blotting results indicated that HIF-1α knockdown increased the expression of the epithelial marker, E-cadherin, and reduced the expression of ZEB1, which is a transcriptional repressor of E-cadherin [24] (Fig. 3C). Since ZEB1 contains HRE sites on its promoter, its expression can be regulated by HIF-1α [17]. To determine whether ZEB1 was downregulated via the knockdown of HIF-1α, we examined the mRNA levels of ZEB1 using qRT-PCR and observed a significant reduction (Fig. 3D). Therefore, HIF-1α upregulated ZEB1, thereby inducing EMT in H1975 cells, and this process was reversed upon knocking down HIF-1α.

Figure 3. Knockdown of HIF-1α inhibits invasion and migration of H1975 cells.
(A) Cells were transfected with siHIF-1α for 48 h. Cell viability was measured using the CCK-8 assay. Statistical analysis was performed using Student’s t-test. Error bars present the mean ± standard deviation (n = 6). ***p < 0.001 vs. siCON. (B) Transwell assays were performed by incubating H1975 cells for 36 h under normoxic conditions after siHIF-1α treatment. Representative images of cell migration and invasion captured under a microscope. Statistical analysis was performed using one‐way ANOVA. Error bars present the mean ± standard deviation (n = 3). ***p < 0.001 vs. siCON. (C) Cells were transfected with siHIF-1α for 48 h. The HIF-1α, E-cadherin, and ZEB1 expression levels were measured using western blotting. GAPDH was used as a loading control. Quantitation of protein intensity was performed using the ImageJ software. Statistical analysis was performed using Student’s t-test. Error bars present the mean ± standard deviation (n = 3). ***p < 0.001 vs. siCON. (D) qRT-PCR analysis of ZEB1. Cells were transfected with siHIF-1α for 48 h. The expression value was normalized to the 18s rRNA level. Statistical analyses were performed using the Student’s t-test. Error bars present the mean ± standard deviation (n = 3). ***p < 0.001 vs. siCON. HDAC6, histone deacetylase 6; HIF-1α, hypoxia-inducible factor 1 alpha; ZEB1, zinc finger E-box-binding homeobox 1; siHIF-1α, small interfering RNA against HIF-1α; GAPDH, glyceraldehyde 3-phosphate; siCON, negative control siRNA.

ACY-241 treatment suppressed EMT in H1975 cells

As the reduction in HIF-1α level suppressed the migration and invasion of H1975 cells, we evaluated the effect of ACY-241 treatment on the migration and invasion of these cells. The wound healing assay showed that ACY-241 treatment reduced the migration ability of cells compared to that of the control. Notably, a significant difference in migration distance was observed after treatment with 2.5 µM ACY-241 (Fig. 4A). Consistent with this finding, ACY-241 treatment significantly reduced the number of migrating and invading cells in the Transwell in a dose-dependent manner (Fig. 4B). Western blot analysis confirmed that ACY-241 treatment led to a dose-dependent decrease in HIF-1α and ZEB1 levels and an increase in E-cadherin expression level (Fig. 4C). Furthermore, ACY-241 treatment significantly decreased the ZEB1 mRNA level (Fig. 4D). Taken together, our data suggest that inhibition of HDAC6 activity by ACY-241 treatment attenuated the migration and invasion of lung cancer cells by reducing HIF-1α level (Fig. 4E).

Figure 4. ACY-241 inhibits migration and invasion in H1975 cell lines.
(A) Wound healing assay of H1975 cells treated with different concentrations of ACY-241. Microphotographs of the wounded area were taken at 0, 24, and 36 h to observe cell migration into the wounded area. Statistical analysis was performed using one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 6). ***p < 0.001 vs. CON. (B) Transwell assays were performed using different concentrations of ACY-241 for 36 h. Representative images of cell migration and invasion were captured under a microscope. Statistical analysis was performed using one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 3). ***p < 0.001 vs. CON. (C) Cells were treated with ACY-241 for 48 h, after which the protein levels of HDAC6, HIF-1α, E-cadherin, and ZEB1 were monitored via Western blot analysis. GAPDH was used as a loading control. Quantitation of protein intensity was performed using the ImageJ software. Statistical analysis was performed using one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 3). **p < 0.01; ***p < 0.001 vs. CON. (D) qRT-PCR analysis of ZEB1. Cells were treated with ACY-241 for 48 h. The expression value was normalized to the 18s rRNA level. Statistical analyses were performed using the one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 3). **p < 0.01; ***p < 0.001 vs. CON. (E) Under normoxic conditions, cancer cells with higher expression of HDAC6 have been shown to increase the stability of HIF-1α protein. The stabilized HIF-1α induces the expression of ZEB1, which decreases the expression of E-cadherin, leading to increased invasion and migration of cancer cells (left). Conversely, reducing HDAC6 through HDAC6 knockdown, or inhibiting HDAC6 activity with ACY-241, decreases the protein level of HIF-1α, which in turn leads to a reduction in epithelial-mesenchymal transition (right). HDAC6, histone deacetylase 6; HIF-1α, hypoxia-inducible factor 1 alpha; ZEB1, zinc finger E-box-binding homeobox 1; GAPDH, glyceraldehyde 3-phosphate ; CON, control.

In this study, we confirmed that ACY-241, a selective HDAC6 inhibitor, plays a crucial role in suppressing EMT by reducing the HIF-1α protein expression level. We confirmed that both knockdown and ACY-241-mediated pharmacological inhibition of HDAC6 reduced the HIF-1α level, which subsequently downregulated ZEB1, leading to reduced migration and invasion of H1975 cells. These findings highlight the pivotal role of HIF-1α regulation via HDAC6 inhibition in the EMT process and suggest the potential significance of ACY-241 treatment in cancer therapeutic strategies.

Under normoxic conditions, we observed that HDAC6 inhibition decreased the HIF-1α protein level (Fig. 1B, C). Typically, in the presence of oxygen, prolyl hydroxylase induces HIF-1α hydroxylation, which is known to induce its proteasomal degradation mediated by the E3 ubiquitin ligase, von-Hippel Lindau tumor suppressor [25]. However, the normoxic expression of HIF-1α in certain cancer cell lines was increased (Fig. 1A). Therefore, different mechanisms can regulate HIF-1α stability, with epigenetic regulation being a notable one. Epigenetic modifiers including DNA and histone methylation as well as histone and non-histone acetylation that affect post-translational modifications have been reported to affect HIF-1α activity and stability [26]. HDACs, a class of enzymes that regulate epigenetic regulation, are key modulators that directly or indirectly affect HIF-1α stability [27]. For example, HDAC1 enhances HIF-1α stability via its deacetylation activity in breast cancer cells [28]. Similarly, HDAC2, HDAC4, and HDAC5 can modulate HIF-1α stability via deacetylation [28,29]. HDAC6 can indirectly modulate HIF-1α stability by regulating the acetylation of HSP70 and HSP90, which are known client proteins of HIF-1α [30]. Conversely, the deacetylation of specific residues on HIF-1α by HDACs may suppress its stability, thereby producing a contrasting outcome [31].

In the present study, cancer cells showing increased HDAC6 expression exhibited an increased HIF-1α expression under normoxic conditions (Fig. 1A). Additionally, HDAC6 knockdown and ACY-241 treatment reduced the HIF-1α level (Fig. 1B and 2B). Therefore, HDAC6 plays a role in maintaining the stability of HIF-1α under normoxia. However, further studies are needed to elucidate the specific mechanism via which HDAC6 inhibition decreases HIF-1α level. The pharmacological inhibitory effects of HDAC6 on regulation of HIF-1α stability have been explored in several studies. Specifically, tubastatin A downregulates HIF-1α by inhibiting transforming growth factor beta 1-induced Akt phosphorylation, thereby attenuating pulmonary fibrosis [32]. Furthermore, tubastatin A enhances HSP90 acetylation in nucleus pulposus cells, which disrupts the formation of the Hsp90/HDAC6 complex and subsequently reduces HIF-1α transcriptional activity [33]. Another HDAC6 selective inhibitor, TC24, can reduce HIF-1α and vascular endothelial growth factor levels by increasing the phosphorylation of cAMP response element-binding protein, thereby enhancing cell cycle arrest and apoptosis in gastric cancer cells [10]. Although efforts to modulate HDAC6 activity for controlling HIF-1α stability are ongoing, research on the use of selective HDAC6 inhibitors in cancers remains limited. ACY-241, a second-generation HDAC6 selective inhibitor, is either undergoing or has completed phase 1 clinical trials for treating advanced solid tumors (NCT02551185), NSCLC (NCT02635061), and multiple myeloma (NCT02400242). In the present study, we demonstrated that ACY-241 treatment in lung and breast cancer cells reduced the increased HIF-1α protein level under normoxic conditions (Fig. 2B). This is the first study to show that ACY-241, a selective HDAC6 inhibitor, can effectively inhibit HIF-1α stability in normoxia.

HDAC inhibitors are considered as effective therapeutic agents for regulating EMT and are currently under investigation in various preclinical studies, either alone or in combination with other anticancer drugs. However, depending on the type of HDAC inhibitor used and type of cancer, EMT may be enhanced, which limits its use in cancer treatment. In particular, suberoylanilide hydroxamic acid, one of the most extensively studied HDAC inhibitors, can promote cell migration and invasion in various cancers, including lung, liver, and prostate cancer [34-36]. This is considered as a side effect of pan-HDAC inhibitors, and is thought to occur owing to their ability to simultaneously inhibit several HDACs [37]. To overcome these challenges, it is necessary to use selective HDAC inhibitors that do not exhibit side effects, such as the promotion of EMT. In our study, ACY-241-treated H1975 cells demonstrated a significant reduction in cell viability (Fig. 2A). Furthermore, ACY-241 treatment reduced the HIF-1α level, which was associated with decreased expression of ZEB1 and E-cadherin (Fig. 4C). Additionally, the altered expression of these targets was associated with a decrease in cell invasion and migration (Fig. 4A, B). In conclusion, ACY-241 can suppress EMT and reduce cell viability in H1975 cells, demonstrating its potential in treating cancer.

None.

This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. NRF-2016R1A6A1A03011325) and Ministry of Science and ICT (MSIT) (No. NRF-2022R1A2C1008787).

The authors declare no conflicts of interest.