HR23A-knockdown lung cancer cells exhibit epithelial-to-mesenchymal transition and gain stemness properties through increased Twist1 stability

BBA - Molecular Cell Research 1866 (2019) 118537

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HR23A-knockdown lung cancer cells exhibit epithelial-to-mesenchymal transition and gain stemness properties through increased Twist1 stability

T

Chung-Yun Yua,1, Bang-Hung Liua,1, Shao-Yi Tanga, Ruei-Yue Lianga, Keng-Hao Hsub, ⁎ Show-Mei Chuanga, a b

Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan Bachelor Program of Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan

A R T I C LE I N FO

A B S T R A C T

Keywords: HR23A EMT Cancer stemness Twist1 Ubiquitination

The epithelial-mesenchymal transition is a major cause of cancer metastasis, and deregulation of the transcription factor, Twist1, is a critical molecular event in the epithelial-mesenchymal transition. The importance of Twist1 protein turnover in this process has not yet been defined. Here, we show that HR23A directly targets the Twist1 protein without changing its gene transcription. Our experiments reveal that: HR23A interacts with Twist1, and this promotes the ubiquitin-mediated proteasomal degradation of Twist1. Depletion of HR23A enhances Twist1 protein levels, epithelial-mesenchymal transition, cancer cell migration and various cancer stemness properties, including the expression of major pluripotency factors, the capacity for tumor-sphere formation in culture and the expression of cancer stem cell surface markers. The increases of these stemness properties are reversed by ectopic expression of HR23A or further knockdown of Twist1 in HR23A-depleted cells. Thus, HR23A-knockdown cells appear to undergo epithelial-mesenchymal transition and take on certain attributes of cancer stemness. Together, our findings indicate that HR23A importantly contributes to regulating Twist1 protein stability, and suggest that altering the stability of Twist1 by modulating HR23A may be a new avenue for therapeutic intervention in cancer.

1. Introduction During tumor progression, the epithelial-to-mesenchymal transition (EMT) contributes to the spreading of tumor cells from the primary site to distant organs (i.e., metastasis). This transition is activated by EMT transcription factors (EMT-TFs), such as Slug, Snail, Zeb1 and Twist, which suppress several epithelial marker genes. At the same time, mesenchymal marker genes are up-regulated. The epithelial cells become polarized, gradually lose their cell-cell contacts and acquire the motility and migratory properties of mesenchymal cells [1,2]. Twist1 is a basic helix–loop–helix (b-HLH) transcription factor that acts as a master EMT-TF and was originally identified as a regulator of mesodermal development [3,4]. The overexpression of Twist1 in various advanced cancers contributes to inducing EMT, which is correlated with higher cancer aggressiveness and poor survival rates [3,5]. Mechanistically, Twist1 represses E cadherin expression by binding the Ebox element, and thereby induces EMT and metastasis [3,6]. Moreover, up-regulation of Twist1 accounts for breast cancer metastasis and is associated with the acquisition of cancer stemness properties, which are

highly correlated with cancer initiation, maintenance, metastasis and chemoresistance [7,8]. Several mechanisms are known to up-regulate Twist1 protein expression during tumor progression and migration. Various studies have identified signals that activate Twist1 transcription and modulate its mRNA translation [5]. For example, TNF-α rapidly induces Twist1 mRNA and protein expression levels to promote cancer invasion [9]. Activation of extracellular signal regulated kinase 1/2 signaling elevates Twist1 mRNA/protein expression in in melanoma cell lines [10]. In a series of MCF-10A-derived cell lines, Nairismagi et al. found that miR-580, CPEB1 and CPEB2 negatively regulate Twist1 expression [11]. Despite this knowledge base in the gene transcription of Twist1, however, few studies have examined its post-translational regulation and the underlying mechanisms. Twist1 was shown to be degraded through the ubiquitin-proteasome system (UPS) [12], and the E3 ubiquitin ligase F-box and leucine-rich repeat protein 14 (FBXL14) is known to target Twist1 for ubiquitin-mediated proteolysis [13]. Thus, Twist1 is a labile protein that can be post-translationally regulated and degraded by ubiquitination. Zhong et al. further demonstrated that

⁎

Corresponding author at: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan. E-mail address: [email protected] (S.-M. Chuang). 1 C-Y Yu and B-H Liu contributed equally to this work. https://doi.org/10.1016/j.bbamcr.2019.118537 Received 15 August 2018; Received in revised form 16 July 2019; Accepted 24 August 2019 Available online 02 September 2019 0167-4889/ © 2019 Elsevier B.V. All rights reserved.

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inhibition of IKKβ prevents Twist1 degradation through SCFβ-TRCP ubiquitination, indicating that SCFβ-TRCP functions as an E3 ligase for Twist1, and that IKKβ-mediated phosphorylation promotes the SCFβTRCP -mediated ubiquitination and degradation of Twist1 [14]. Members of the Rad23 family of ubiquitin-like ubiquitin-associated (UbL-UBA) domain-containing proteins reportedly play dual roles in nucleotide excision repair (NER) and promoting the ability of the proteasome to recognize a ubiquitinated substrate for degradation [15]. HR23A and HR23B are the human orthologs of the yeast Rad23 (HR23) proteins; these proteins possess four domains that mediate interactions with ubiquitin, the proteasome and/or various proteins: an N-terminal ubiquitin-like (UbL) domain, an XPC-binding domain (XPCB) and two ubiquitin-associated (UBA) domains comprising an internal UBA1 domain and a C-terminal UBA2 domain. The UbL domain of Rad23 is important for its interaction with the 26S proteasome and participation in the UPS [15]. The UBA domain was originally identified as a sequence motif that non-covalently binds to polyubiquitin chains [16]. The XPC-binding domain is required for the association with XPC, which is important for recognizing UV-damaged DNA, and is essential for NER. The so-called shuttle vector model proposes that polyubiquitinated proteins are recognized and delivered by Rad23 family members and then further recognized by the 26S proteasome, a multicatalytic complex [16]. However, there is long-standing debate regarding which proteins recognize polyubiquitinated substrates and deliver them to the proteasome for degradation. Although HR23A and HR23B were found to be functionally interchangeable in a reconstituted NER reaction [17], the requirement for these proteins remains unresolved in mammalian cells. Knockout mouse models have revealed that HR23A and HR23B play fully redundant roles in NER and partially redundant functions in embryonic development [18], suggesting that they may have distinct functions in specific biological processes [18–20]. The distinct functions of HR23A and HR23B are also supported by a report that these proteins act differently in their binding to the proteasome and polyubiquitinated proteins [21]. Furthermore, using a siRNA-based screening to knock down various UBD (ubiquitin-binding domain)-containing proteins in human embryonic kidney (HEK) 293 cells, Fang et al. found that HR23A, but not HR23B, can down-regulate TRAF2 by promoting its degradation through the UPS [22]. This identified a novel function of HR23A in negatively regulating the expression of cytokines triggered by RIG-1/ MDA signaling in antivirus immunity. These studies support the notion that HR23A functions distinctly from HR23B in regulating cellular functions, such as cell cycle progression, immunity and stress responses. In preliminary work, we observed a fibroblast-like morphological change when we knocked down HR23A, but not HR23B, using lentivirus-derived shRNAs in A549 cells. These findings strongly encouraged us to further investigate the role of HR23A in the homeostatic regulation of EMT. Here, we explore the specific function of HR23A by genetically manipulating its gene expression in A549 cells as a tumor model. Our findings demonstrate that HR23A critically regulates Twist1 protein expression, and thereby contributes to cancer EMT and the maintenance of cancer stemness.

transfected with the indicated construct using the jetPEI transfection reagent according to the manufacturer's protocol (Polyplus-transfection SA, Illkirch Cedex, France). IgG sepharose was purchased from GE Healthcare (Piscataway, NJ, USA). The utilized antibodies are listed in Supplemental Table 1. 2.2. Cell culture The A549 human lung adenocarcinoma cell line was purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu City, Taiwan). The CL1-0 human lung adenocarcinoma cells, which were established from a non-small-cell lung carcinoma tumor [24], were gifted by Dr. Jeremy J. W. Chen (Institute of Biomedical Sciences, National Chung Hsing University, Taichung City, Taiwan). Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Invitrogen (Grand Island, NY, USA). A549 and CL1-0 cells were cultured in RPMI medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and were maintained at 37 °C in a humidified incubator containing 5% CO2 in air. 2.3. RNA interference To knock down HR23A and HR23B, A549 cells were infected with lentiviruses carrying HR23A- or HR23B-specific shRNAs (from National RNAi Core Facility, Academia Sinica, Taiwan) according to the manufacturer's recommendations. Infected cells were confirmed by assaying HR23 protein levels. For small interfering RNA (siRNA)-mediated inhibition, ON-TARGET plus SMART pools corresponding to HR23A, HR23B and Twist1 siRNAs were purchased from Dharmacon Research (Lafayette, CO, USA). Non-targeting siRNA duplexes were used as the negative control siRNA (Dharmacon Research). Cells were transfected with 25 nM siRNAs using Lipofectamine RNAiMAX (Invitrogen) in glucose-free Opti-MEM (Invitrogen) according to the manufacturer's recommendation. 2.4. Two-dimensional electrophoresis Cell extracts were prepared in rehydration buffer (8 M urea, 0.5% CHAPS, 0.2% DTT, 0.002% bromophenol blue and 0.5% IPG buffer; immobilized polyacrylamide gel buffer, pH 4 to 7; GE Healthcare) and transferred to 18-cm Immobiline DryStrip gels (pH 4 to 7; GE Healthcare). The protein solutions were allowed to soak into the DryStrips at 20 °C overnight, and the proteins were resolved by isoelectric focusing (IEF). After IEF, the gel strips were rinsed in 20 ml equilibration buffer (2% SDS, 50 mM Tris-Cl, pH 8.8, 6 M urea, 30% glycerol, 0.002% bromophenol blue and 1% DTT) for 15 min, followed by a second wash for 15 min in 20 ml of equilibration buffer lacking DTT and containing 4% iodoacetamide. The strips were then layered onto a 10% SDS-polyacrylamide gel and separated in the second dimension followed by PhastGel Blue R (GE Healthcare) staining. The spots were excised and subjected to protein identification using MALDITOF MS.

2. Materials and methods 2.5. Quantitative real-time PCR (qRT-PCR) 2.1. Chemicals, plasmids and antibodies Total RNAs were extracted from cultured cells using the TRIzol reagent (Invitrogen) and cDNAs were prepared using a Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Specific primers were designed using the Probe Finder software, which is available online at the Universal ProbeLibrary Assay Design Center (Roche Applied Science, Germany) and is based on the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) Guidelines. The probes were obtained from the Universal ProbeLibrary collection (Roche). qPCR analysis was performed utilizing a LightCycler Nano instrument (Roche), and the results

Puromycin was purchased from Invivogen (San Diego, CA, USA). MG132 was purchased from TOCRIS Bioscience (Ellisville, MO, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The full-length cDNA encoding HR23A was amplified [23] and inserted into pCMV-tag-2B and pCMV-Myc to generate pCMV-Flag-HR23A and pCMV-Myc-HR23A, respectively. The full-length cDNA encoding Twist1 was cloned into pcDNA3.1-V5-HisTOPO to generate pcDNA-Twist1-His. All constructs were confirmed by sequencing. For transfection experiments, cells were transiently 2

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agar. The agar-cell mixture was plated atop a bottom layer consisting of a 0.5% agar-medium mixture. After 4 weeks, the colonies were stained with crystal violet and counted.

of each experiment were normalized with respect to the expression of the control housekeeping gene, TBP. 2.6. Wound-healing assay

2.12. Sphere-formation assay Wound-healing assays were performed under microscopic imaging to assess the ability of cell migration. Cells were plated to 6-well plates and grown to confluence. The monolayers were wounded using the tip of a sterile 200-μl pipette. The debris was removed by two washes with PBS, and the cells were incubated with medium containing 0.5% FBS. Bright-field images of randomly selected views along the scraped line were taken under microscopy (Olympus IX71, Japan) at the indicated time points.

Cells were adjusted to 100 cells/200 μl in serum-free DMEM (Invitrogen) supplemented with B27 (Invitrogen), 10 ng/ml human recombinant epidermal growth factor (EGF; BD Biosciences) and 10 ng/ ml basic fibroblast growth factor (bFGF; BD Biosciences), and cultured in ultra-low attachment 96-well plates (3474; Corning, Oneonta, NY, USA). Spheres with a diameter of > 50 μm were counted [7] in each well under inverted contrast microscopy.

2.7. 3D matrigel culture

2.13. Immunofluorescence staining

Cells (1 × 105) were mixed well with 2 mg/ml matrigel (BD Bioscience, San Jose, CA, USA) in serum-free medium and seeded to a 96-well plate. Cell morphology was monitored for the indicated durations under microscopy (Olympus IX71).

Cells grown on cover slides were washed in PBS, fixed in 4% formaldehyde/PBS, washed in PBS, and blocked in 5% bovine serum albumin/PBS. The cells were incubated with primary antibodies (in 5% bovine serum albumin/PBS) overnight at 4 °C and washed. Binding of primary antibodies to N-cadherin and E-cadherin was detected with Rhodamine-conjugated goat anti-mouse (Millipore) and FITC-conjugated goat anti-mouse (Millipore), respectively. Fluorescence images were obtained using an Olympus IX71 fluorescence microscope.

2.8. Transwell migration/invasion assay Transwell inserts (8-μm pore diameter; Millipore, MA, USA) were used to measure the cell migration ability. The lower chambers were filled with medium containing 10% FBS. Cells (5 × 104) were suspended in serum-free medium and seeded to the upper chambers. After 24 h, nonmigrated cells were removed from the upper side of the insert with a cotton swab, and cells on the lower insert surface were fixed and stained with crystal violet. All migrated cells were counted, and the data are presented as migrated cells per insert. Experiments were performed in triplicate. To assess the invasive ability of cells, Transwell inserts were pre-coated with 1 mg/ml matrigel in serum-free medium and placed in a 37 °C incubator for 30 min to allow the gel to solidify, and then the cells were loaded.

2.14. Statistical analysis Data are presented as the mean ± S.D. of at least three independent experiments. The differences between the control and experimental groups were calculated using the SigmaPlot 10.0 software, and the Student's t-test was used to evaluate the levels of significance. The results presented in the graphs are representative of multiple independent experiments; * indicates P < 0.05, which was considered statistically significant. 3. Results

2.9. Luciferase assay 3.1. Knockdown of HR23A induces EMT The previously reported the Twist1-responsive E-box of the E-cadherin promoter (5′-CACCTG-GCTGCT-CACCTG-3′) or a mutant version thereof (5′-acttct-GCTGCT-acttct-3′) [3,25] was cloned into the pGL3promoter plasmid (Promega, Madison, WI, USA) to generate E-box-Luc reporter constructs. Luciferase activity assays were performed according to the manufacturer's instructions (Promega). Briefly, at 24 h post-transfection, the cells were lysed by being scraped into 250 μl lysis buffer. The lysates were cleared by a brief centrifugation, and 50 μg of each lysate was subjected to luminescence measurement using a Sirus Luminometer (Berthold Detection System, Pforzheim, Germany). All samples were assayed in triplicate.

We initially observed that A549 cells subjected to lentivirus-mediated shRNA knockdown of HR23A (sh-HR23A) acquired a fibroblastlike mesenchymal appearance compared with control cells, suggesting they may undergo epithelial-to-mesenchymal transition (EMT). To address this assumption, we performed two-dimensional electrophoresis of lysates from sh-HR23A and sh-control cells, isolated the protein spots and identified the proteins by MALDI-TOF MS. We found that, for example, Vimentin and Cytokeratin 8 were up- and down-regulated, respectively, in sh-HR23A cells compared to sh-control cells (Fig. 1A and Supplemental Fig. S1). Further, analysis of EMT markers revealed that the classic epithelial markers, E-cadherin and Cytokeratin 8, were down-regulated, while the mesenchymal markers, N-cadherin and Vimentin, were up-regulated in si-HR23A (Fig. 1B) and sh-HR23A cells (Fig. 1C) compared with their respective controls. In contrast, overexpression of Flag-HR23A reversed the protein levels in sh-HR23A cells (Fig. 1C). The observed loss of epithelial markers and induction of mesenchymal markers in HR23A-knockdown cells indicated that these cells underwent EMT. Moreover, we found that this was not unique to A549 cells. Increased mesenchymal markers and decreased epithelial markers were also observed in CL1-0 cells subjected to knockdown of HR23A by siRNAs and lentivirus-derived short hairpin RNAs (Supplemental Fig. S2A), indicating that HR23A-knockdown cells acquired a mesenchymal appearance compared with control cells. Although HR23A and HR23B are structurally similar, shRNA-mediated knockdown of HR23B (sh-HR23B) did not have detectable effect on the fibroblast-like morphology of these cells (Fig. 1D and E). Ectopic expression of HR23A reversed the cell morphology of sh-HR23A cells to

2.10. Real-time cell analysis (RTCA) To measure the electrical impedance of touched cells, cells (1 × 104/well) were seeded onto E-plates (Roche, Germany), the plates were placed onto the RTCA station (xCELLigence Real-Time Cell Analysis System, Roche), and the impedance was measured hourly for 6 h. Impedance is presented as the cell index (CI) = (Zi − Z0) [Ohm]/ 15[Ohm], where Z0 is the background resistance and Zi is the resistance at an individual time point. A normalized cell index was determined as the CI at a certain time point (CIti) divided by that at the normalization time point (CInml_time). 2.11. Soft-agar assay for colony formation and anchorage-independent growth Cells (1 × 104) were suspended in culture medium containing 0.3% 3

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in human lung cancer cells.

that of sh-control cells (Fig. 1F and G). A similar HR23A-knockdownrelated cell morphology was observed in another lung adenocarcinoma cell line (CL1-0) (Supplemental Fig. S2B). As the induction of mesenchymal markers and morphological change observed in HR23Aknockdown cells, we suggest that knockdown of HR23A induced EMT

3.2. HR23A regulates the stability of Twist1 EMT is activated by EMT transcription factors (EMT-TFs), such as

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Fig. 1. Knockdown of HR23A increases the expression of EMT. (A) HR23A specifically knocked-down in A549 cells was selected (sh-HR23A) by means of the lentiviral RNA interference system. Total proteins were resolved by isoelectric focusing (IEF) (pH 4 to 7) followed by SDS-PAGE stained with PhastGel Blue R. The spots were identified by MALDI-TOF MS. (B) Knockdown of HR23A increases N-cadherin and Vimentin but decreases E-cadherin and Cytokeratin 8 expression. (C) sh-HR23A cells were transfected with 2 μg empty vector or Flag-HR23A for 24 h followed by immunoblot analysis. (D) The cell morphology was observed by optical microscope and the protein levels of HR23A and HR23B were confirmed by immunoblot analysis (E). (F) (G) The cell morphology was detected in sh-control, shHR23A cells without and with 2 μg Myc-HR23A transfection. 4

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cadherin and up-regulation of N-cadherin on the cell membranes of shHR23A A549 cells, while overexpression of Flag-HR23A reversed the levels of E-cadherin and N-cadherin on cell membranes. Similar results were observed with Twist1-depletion in these sh-HR23A A549 cells (Fig. 3G). The present work is the first to show that HR23A functions to regulate Twist1 protein stability and EMT. Taken together, the results of our ectopic expression and knockdown experiments indicate that the level of HR23A is inversely correlated with the Twist1 protein function. These results also indicate novel roles of HR23A in EMT through modulating Twist1 function.

Twist1, Slug, Snail, and Zeb1 which suppress several epithelial marker genes. At the same time, mesenchymal marker genes are up-regulated. We further found that the protein levels of critical EMT-TFs, including Twist1, Slug and Zeb1, were significantly enhanced by knockdown of HR23A (Fig. 2A). Exogenous expression of HR23A reversed the protein expression levels of Twist1, Slug and Zeb1 in sh-HR23A cells (Fig. 2A and Supplemental Fig. S2C), implying that HR23A may regulate EMT by manipulating the protein levels of these EMT-inducing transcription factors. Of the EMT regulators, Twist1 has been well demonstrated to be a critical causality of EMT. Thus, we investigated the role of HR23A in modulating Twist1 expression and found that HR23A negatively modulates Twist1 turnover using cycloheximide chase assays. Interestingly, the stability of Zeb-1 and Slug were marginally affected in HR23A knockdown cells. We therefore focus on the role of HR23A on regulating Twist1 expression. Fig. 2B indicate that HR23A knockdown increased the stability of Twist1 and rescue of HR23A expression reversed this effect on Twist1 protein levels (Fig. 2B–C and Supplemental Fig. S2D). These findings indicate that HR23A accelerates Twist1 protein turnover. In contrast, the mRNA level of Twist1 was not altered by knockdown of HR23A (Fig. 2D). The effect of HR23A on the Twist1 protein level was blocked by the proteasome inhibitor, MG132 (Fig. 2E–F and Supplemental Fig. S2E), indicating that the UPS is required for the HR23A-mediated down-regulation of Twist1. The HR23 proteins appear to play complex roles in modulating proteasome-mediated proteolysis. Depending on the cellular context, the binding of HR23 proteins to ubiquitylated substrates may increase or decrease the proteasomal degradation of these substrates: HR23 proteins can bind to and escort polyubiquitylated proteins to the proteasome, increasing their degradation or, conversely, they may protect target proteins from proteasomal degradation, possibly by interfering with ubiquitin chain elongation or the proteasome-mediated proteolysis of ubiquitylated proteins. Indeed, Twist1 poly-ubiquitination was significantly attenuated upon knockdown of HR23A (Fig. 2G and Supplemental Fig. S2F). Since HR23A plays a critical role in the UPS, we hypothesized that HR23A, which is a poly-ubiquitin chain carrier, might associate with Twist1 and deliver it to the 26S proteasome for degradation. To examine this possibility, we immunoprecipitated endogenous HR23A and performed immunoblot analysis. As shown in Fig. 2H, endogenous Twist1 coprecipitated with HR23A. This interaction was recapitulated when we performed immunoprecipitation of Twist1 (Fig. 2I), suggesting that HR23A interacts with Twist1 in vivo. Taken together, our results indicate that under HR23A depletion, Twist1 undergoes less proteasomal degradation and is thereby upregulated at the protein level.

3.4. Knockdown of HR23A promotes cancer cell stemness in vitro In addition to the involvement of EMT-TFs in cancer initiation and metastasis, recent studies have indicated that these transcription factors also mediate cancer cell proliferation, survival, drug resistance and cancer stemness properties [26]. Representative works by Mani et al. [7] and Morel et al. [8] demonstrated that the induction of EMT-TFs in an immortalized mammary epithelial cell line causes these cells to not only undergo EMT but also to acquire stemness properties, such as upregulation of stem-cell markers and the ability to efficiently form mammospheres, soft agar colonies and tumors. As Twist1-induced EMT promotes cancer stemness [7,8] and HR23A depletion enhances the Twist1 protein level in A549 cells, we examined whether knockdown of HR23A could enhance stem cell-like phenotypes in A549 cells. Interestingly, HR23A-knockdown cells displayed a higher expression of cancer stem cell surface marker CD44 as compared with sh-control cells (Fig. 4A). In addition to regulating the pluripotency of embryonic stem cells, the stemness-associated transcriptional factors, OCT3/4, SOX2, Kruppel like factor 4 (KLF4) and Myc, are also expressed in cancer cells, where they play critical roles in maintaining stemness properties. Previous studies have noted that over-expression of major pluripotency factors is significantly correlated with a higher histological grade, poor patient survival and an increased capacity for tumor-sphere formation in culture and xenograft models [27,28]. As these results suggested that cancer stemness properties are increased in sh-HR23A cells, we examined the molecules relative to stem cell properties in sh-HR23A cells. We found that the pluripotency factors, OCT4, Sox2, c-Myc, and Bmi1, which act as downstream effectors of Twist1 in maintaining cancer stemness, were up-regulated in HR23A-knockdown cells (Fig. 4B). Ectopic expression of Flag-HR23A decreased the levels of stemness-associated transcription factors (Fig. 4B). Furthermore, knockdown of HR23A enhanced anchorage-independent growth, and the over-expression of Flag-HR23A reversed this up-regulated anchorage-independent growth in sh-HR23A cells (Fig. 4C). Most importantly, spheroid-forming ability, as characterized by increased sphere sizes and numbers, was enhanced by knockdown of HR23A relative to control cells. Again, ectopic expression of Flag-HR23A also reversed the acquisition of these stemness properties, as evidenced by decreases in the levels of stemness-associated transcription factors, anchorage-independent growth, spheroid-forming ability (Fig. 4B–D). These results indicated that in addition to inducing mesenchymal migration/invasion and EMT, knockdown of HR23A also enhances the stemness properties of A549 cells. To clarify whether HR23A knockdown induced cancer cell stemness properties by modulating Twist1, the expression of Twist1 was depleted in sh-HR23A cells. Indeed, sphere-forming assays revealed that the numbers and sizes of the spheroids decreased markedly following Twist1 knockdown (Fig. 4D). Furthermore, Twist1 knockdown reversed the protein expression of stemness-associated transcription factors in sh-HR23A cells (Fig. 4E and Supplemental Fig. S3A). Taken together, these findings indicate that the HR23A knockdown-induced enhancements of EMT and cancer stemness properties were rescued by the down-regulation of Twist1.

3.3. Knockdown of HR23A suppresses E-cadherin expression Twist1 is the most thoroughly studied EMT-TF. Given that it is known to transcriptionally suppress E cadherin, we next investigated whether depletion of HR23A modulated the transcription activity of Twist1. Indeed, the mRNA level of E cadherin was significantly downregulated (Fig. 3B) while that of N-cadherin was up-regulated (Fig. 3C) in sh-HR23A cells relative to sh-control cells. Next, we inserted the Twist1-responsive E-box of the E-cadherin promoter [3,25] into the pGL3-promoter vector upstream of a luciferase reporter and performed luciferase assays in sh-control and sh-HR23A cells. Indeed, the luciferase activity was suppressed in sh-HR23A cells relative to sh-control cells, and this suppression was released by restoring HR23A expression in sh-HR23A cells (Fig. 3D). In contrast, a reporter construct harboring a mutated E-box element exhibited a very limited response upon HR23A depletion (Fig. 3E). Silencing of Twist1 in sh-HR23A cells significantly induced the luciferase activity, as compared to that in shHR23A cells transfected with a scrambled control (Fig. 3F), indicating that the effect of HR23A on the E box element is Twist1-dependent. Immunofluorescence staining also revealed down-regulation of E5

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Fig. 2. HR23A regulates the protein stability of Twist1. (A) sh-HR23A cells were transfected with 2 μg empty vector or Flag-HR23A 2 for 24 h followed by immunblot analysis to determine the levels of EMT transcription factors. (B) Cells were left treated with 50 μg/ml cycloheximide (CHX) for 0–2 h. The levels of Twist1 were determined by immunoblot analysis. (C) The levels of Twist1 in cells were determined by qRT-PCR. (D) sh-HR23A cells were transfected 2 μg empty vector or MycHR23A for 24 h followed by treatment with CHX for 0–2 h. The levels of Twist1 and Myc-HR23A were measured by immunoblot analysis. (E) (F) Cells were treated with or without 10 μM MG132 for 1 h. The levels of Twist1 were detected by immunoblot analysis. (G) Cells were transfected with 2 μg Twist1-His for 24 h. Cell lysates were prepared and Twist1-His proteins were precipitated by Nickle sepharose. The ubiquitin chains on Twist1 were detected by immunoblot analysis. (H) Endogenous HR23A was immunoprecipitated by anti-HR23A and the interacted Twist1 was detected by immunoblot analysis. (I) endogenous Twist1 was immunoprecipitated by ant-Twist1 and associated HR23A was detected by immunoblot analysis.

relative to sh-control cells (Fig. 5E).

3.5. Knockdown of HR23A enhances cell migration and invasion ability Through EMT, the epithelial cells acquire the motility and migratory properties of mesenchymal cells. Thus, we next investigated whether HR23A knockdown could enhance cell migration. As expected, HR23A-knockdown in A549 cells were more mobile than control cells in wound-healing assays (Fig. 5A and B). HR23A knockdown was also found to increase the cell migration of CL1-0 cells (Supplemental Fig. S3B). This increased cell migration ability was significantly rescued by expression of Myc-tagged HR23A (Fig. 5C). Transwell migration assays also confirmed the higher mobility of sh-HR23A compared to sh-control cells (Fig. 5D). Moreover, our in vitro matrigel invasion assay further supported the notion that sh-HR23A cells show increased mobility

A

3.6. HR23A modulates EMT and cell migration through Twist1 To confirm that the effects of HR23A knockdown on cell motility are mediated by Twist1, we knocked down Twist1 expression in sh-HR23A cells. Immunoblotting with specific antibodies confirmed that Twist1 was efficiently depleted in sh-HR23A cells, and that this was accompanied by up-regulation of E-cadherin and Cytokeratin 8 and downregulation of N-cadherin and Vimentin (Fig. 6A and Supplemental Fig. S3C). Twist1 knockdown significantly reduced the cell migration ability of sh-HR23A cells, as assessed by Transwell migration assays and wound-healing assays (Fig. 6B and C).

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Fig. 3. HR23A regulates the expression of EMT markers. The mRNA level of HR23A (A), E-cadherin (B) and N-cadherin (C) in sh-C cells and sh-HR23A cells were determined by qRT-PCR. (D) The cells sh-control, sh-HR23A cells without and with Myc-HR23A expression were transfected with the E box-Luc reporter construct for 2 days. Cells were lysed for the analysis of the luciferase activities. (E) Mutant E-box was replaced to perform the luciferase assay. (F) sh-HR23A cells were transfected with the E box-Luc reporter construct illustrated together with the si-Twist1 or the si-control. Cells were lysed 2 days later for the analysis of the luciferase activities. (G) sh-HR23A cells were transfected with 2 μg empty vector, Flag-HR23A or 30 nM si-Twist1 for 24 h followed by immunofluorescence staining to measure the levels of E-cadherin and N-cadherin on cell membranes. The images were acquired by IX-71 fluorescence microscope (Olympus, Tokyo, Japan). 7

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Fig. 4. Knockdown of HR23A induces stem-like properties. (A) The cells sh-control and sh-HR23A were subjected to flow cytometry analysis to measure the expression of CD44. (B) The protein levels of stemness-associated transcriptional factors OCT4, SOX2, Myc and Bmi1 were determined in sh-control, sh-HR23A cells without and with Flag-HR23A expression. (C) Soft agar colony formation by sh-control, sh-HR23A cells without and with Flag-HR23A expression was carried out to measure anchorage-independent growth. The panels shown to the upper are representative results of colonies formed by sh-control, sh-HR23A cells without and with Flag-HR23A expression. The histogram shown to the bottom indicated the numbers of colonies. The results represent the mean ± S.D. of four independent experiments. (D) Sphere-formation assay. The panels shown to the upper are representative results of spheres formed by sh-control, sh-HR23A cells, sh-HR23A with Flag-HR23A expression or si-Twist1. Scale bar, 50 μm. The histogram shown to the bottom indicated the numbers of spheres > 50 μm in diameter. The results represent the mean ± S.D. of four independent experiments. (E) The cells sh-control, sh-HR23A, sh-HR23A with si-Twist1 were subjected to immunoblot analysis to detect the levels of stemness-associated transcriptional factors.

HR23A knockdown alters cell phenotypic outcomes in both two- and three-dimensional culture. Adhesion assays also showed that HR23Aknockdown cells exhibited a decreased adhesion ability, as assayed using an xCELLigence system over 5 h (Fig. 7D). Based on the results of our functional assays, we conclude that lung cells subjected to knockdown of HR23A undergo EMT and take on certain attributes of cancer stemness. These data support our hypothesis that HR23A regulates tumorigenesis by modulating Twist1 protein turnover.

The ability of cancer cells to expand outward from an initial spheroid into surrounding 3D collagen, which mimics the in vivo collagen-rich dermal layer, is a mark of their invasive capacity [29,30]. The A549 cell line is noninvasive and displays a spheroid phenotype in 3D culture [31]. Strikingly, sh-HR23A cells displayed increased spheroid outgrowth compared with control cells (Fig. 7A). Whereas shcontrol exhibited a cobblestone-like morphology, sh-HR23A cells displayed a spike-like structure reminiscent of a metastatic phenotype. We observed the same phenotypic change when we used siRNAs to knock down HR23A, with si-control cells showing a spheroid-like structure, while HR23A-knockdown cells exhibited a sea-urchin-like morphology (Fig. 7B). Overexpression of Myc-HR23A restored the spike-like structure to a spheroid-like morphology (Fig. 7C). These results suggest that

4. Discussion In the emerging picture, both HR23 proteins play important roles in 8

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Fig. 5. Knockdown of HR23A enhances cell migration ability in A549 cells. (A) After serum starvation, wound healing assays were carried out. The cell images were acquired by optical microscope for 24 h. (B) A549 cells were transfected with 30 nM si-RNA against HR23A for 24 h followed by serum starvation for another 24 h. Wound healing assays were then carried out. The images were acquired by optical microscope for 24 h. (C) sh-HR23A cells were transfected with 2 μg empty vector or Myc-HR23A for 24 h followed by serum starvation for another 24 h. The cell images were acquired by optical microscope. (D) Transwell assays were applied to measure cell migration (D) and invasion (E) ability in control and HR23A-knockdown cells.

correlation of Twist1-mediated EMT with cancer stemness is still a controversial issue in the field of tumorigenesis [35]. Recent studies have shown that EMT is a dynamic and reversible process during tumorigenesis and metastasis, and that it may not be linked to the maintenance of cancer stemness properties. Using conditional deletion of Twist1 at different stages of skin tumorigenesis, Beck et al. showed that Twist1 is essential for tumor maintenance. The authors further showed that Twist1 inhibits oncogene-induced apoptosis in a p53-dependent manner but promotes tumor cell proliferation and propagation via a p53-independent mechanism, and that the ability of Twist1 to confer cancer stem cell properties during tumor initiation is independent of its role in EMT during tumor invasion [36]. The same conclusion was also drawn by Schmidt et al., who found that Twist1 induced EMT without conferring any mammosphere-forming ability, and that the subsequent withdrawal of Twist1 yielded robust mammosphere-forming abilities despite the cells having reverted to an epithelial morphology [37]. Although our present findings support the idea that Twist1 promotes EMT-derived cancer stemness, the relationship between EMT and stemness still warrants further investigation. The cancer stem cell phenotype is associated with a reduced sensitivity to chemotherapeutic agents. Cells of various cancer types with phenotypic EMT changes or overexpression of certain EMT-TFs reportedly exhibit chemoresistance against various therapeutic agents, including oxaliplatin, tamoxifen, gefitinib, gemcitabine and paclitaxel [38–42]. Some evidence suggests that Twist1 enhances the acquired drug resistance of cancer cells [43,44]. Our previous reports indicated that knockdown of HR23A enhances the resistance of A549 cells to

NER and proteolysis, but HR23A and HR23B play individual roles in distinct cellular processes. In our search to determine the distinct functions of HR23A, we found that HR23A-knockdown lung cell lines possess higher mobility and mesenchymal phenotypes. By combining morphological assessment, biological marker data and functional analyses performed in vitro and in vivo, we herein show that HR23A modulates Twist1 protein stability and consequently regulates its function. Knockdown of HR23A enhances the protein level of Twist1, thereby promoting EMT and stemness properties, the latter of which are associated with a more potent motile capacity and the resistance of cancer cells to anticancer drugs [32]. This work introduces a unique function of HR23A that is distinct from those of HR23B, and also elucidates a novel mechanism for regulating Twist1 protein turnover. In addition to the involvement of EMT-TFs in cancer initiation and metastasis, recent studies have indicated that these transcription factors can also trigger cancer cell proliferation, survival, senescence, drug resistance and the acquisition of cancer stemness properties [26]. Among the EMT-TFs, Twist1 promotes EMT, invasion and cancer stem cell properties, indicating that EMT and cancer stemness are mechanistically linked. The molecular mechanisms linking Twist1-mediated EMT to cancer stemness remain unknown. Yang et al. showed that Twist1 directly binds the Bmi1 promoter region and regulates the expression of this stemness factor, and that both proteins are required for the induction of EMT and stemness in head and neck squamous cell carcinoma (HNSCC) [33]. Twist1 was also shown to promote the generation of a breast cancer stem cell phenotype by down-regulating CD24 expression in human breast cancer cell lines [34]. However, the 9

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without altering Twist1 mRNA expression. Moreover, the same study showed that Ser 68 phosphorylation is positively correlated with the levels of Twist1 protein and JNK activity in invasive human breast ductal carcinomas and in progesterone receptor-negative and HER2positive breast cancers [49]. Finally, a study showed that Twist1 stability is increased by interleukin 6 stimulation, which triggers the casein kinase 2-mediated phosphorylation of Twist residues S18 and S20, thereby inhibiting its degradation [50]. Based on our present data and the previous reports, we reason that under normal conditions HR23A regulates the dynamic turnover of Twist1 to suppress cell motility and modulate cell proliferation. Dysregulation of Twist1 by changes in either gene expression or protein turnover, however, leads to tumor progression and metastasis. Future work is needed to examine the detailed mechanism responsible for regulating the HR23A-Twist1 interaction in normal and tumor cells. Interestingly, the autophagy regulator, p62, has been shown to bind to Twist1 and prevent its proteasomal degradation, increasing EMT, in vitro invasiveness and in vivo metastasis [51]. Autophagy could also maintain tumor stem cell quiescence to enhance drug resistance [52]. We previously demonstrated that the chemotherapeutic drug, cisplatin/ oxaliplatin, induced autophagy in lung cancer cell lines with knockdown of HR23A [32] and that this, in turn, protected cancer cells from

DNA-damaging agents by enhancing the stability of Chk1 [23,45]. In the present study, we further show that knockdown of HR23A enriches the population of lung cancer stem cells by up-regulating the protein expression of Twist1, which participates in maintaining their stemness. This would make the cells more resistant to chemotherapy, which is consistent with previous findings [7]. Based on the existing evidence and our present results, we hypothesize that HR23A normally regulates Twist1 protein turnover to suppress cell transformation. Dysregulation of HR23A may cause up-regulation of Twist1 protein expression, leading the cells to acquire tumorigenicity, stemness properties and metastatic capabilities. Moreover, we propose that EMT-TF expression levels in human tumor biopsies may act as predictive markers that could enable the stratification of therapy-sensitive and therapy-resistant subgroups. Post-translational mechanisms have also been demonstrated to modulate the expression and function of EMT-TFs. For example, the phosphorylation, glycosylation and oxidation of Snail have been shown to regulate its proteasomal degradation, stabilization and nuclear translocation [46–48]. Many different signaling pathways regulate Twist1 expression. Activation of mitogen-activated protein kinase signaling by active Ras protein or TGF-β treatment was shown to significantly increase Ser 68 phosphorylation and Twist1 protein stability 10

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apoptosis. In conjunction with these previous results, our present findings lead us to conclude that dysregulation of HR23A attenuates cell growth, induces autophagy and enhances Twist1 stability, which further enhances the stemness and resistance to DNA damaging agents in cancer cells. The current study therefore enhances our understanding of the mechanisms responsible for regulating Twist1 expression in cancer cells and might open new therapeutic avenues for treating cancer. Our results also suggest that HR23A might play a critical role in controlling the population and autophagic activity of cancer stem cells. If we connect the Twist1-EMT related processes and the regulation of autophagy with the dual functions of HR23A, we might hypothesize that dysregulation of the HR23A-Twist1 axis alters cancer cell survival and drug resistance, shifting cancer cells towards the formation of aggressive drug-resistant carcinomas. In contrast with the relatively well understood role of Twist1, the role of HR23A in cancer is still mysterious. Our analysis of data available online revealed that the mutation status of HR23A is not correlated with the survival rate in human cancers, based on the TCGA database (https://cancergenome.nih.gov/) and the online survival analysis tool, Kaplan-Meier Plotter (http://kmplot.com/analysis/). Moreover, the information provided by Expression Atlas at the European Bioinformatics Institute (EMBL-EBI) revealed that HR23A expression is not correlated with tumorigenesis: HR23A is expressed at a low level in breast carcinoma, osteosarcoma and non-small-cell lung carcinoma, but at a higher level in lung squamous cell carcinoma and non-melanoma skin squamous cell carcinoma [53](https://cancergenome.nih.gov/). We speculate that the functions of HR23A might be differentially altered in different cancers and at different stages during tumorigenesis. Further investigations will be needed to elucidate the detailed mechanisms through which HR23A regulates cancer progression. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbamcr.2019.118537.

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Author contributions CYY, KHH and SYT carried out the 2D electrophoresis, immunoblotting, qRT-PCR, cell migration/invasion assays and luciferase assays with the assistance of RYL. RYL constructed the luciferase reporters. BHL performed the identification of stem cell markers by immunoblotting, flow cytometry, sphere-forming assay and immunofluorescence staining. SMC conceived the study, participated in designing and coordinating the study, and contributed to writing, reviewing, and/or revising the manuscript. All authors revised and approved the final version of submitted manuscript. Transparency document The Transparency document associated with this article can be found, in online version. Declaration of competing interest None of the authors has any conflict of interest or other relationship/activity that could appear to have influenced the submitted work. Acknowledgements This work was supported by a grant from the Ministry of Science and Technology, Taiwan (MOST 107-2320-B-005-016). References [1] J.P. Thiery, Epithelial-mesenchymal transitions in tumour progression, Nat. Rev. Cancer 2 (2002) 442–454. [2] B. De Craene, G. Berx, Regulatory networks defining EMT during cancer initiation and progression, Nat. Rev. Cancer 13 (2013) 97–110.

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