TIP60 is required for tumorigenesis in non‐small cell lung cancer

Abstract Histone modifications play crucial roles in transcriptional activation, and aberrant epigenetic changes are associated with oncogenesis. Lysine (K) acetyltransferases 5 (TIP60, also known as KAT5) is reportedly implicated in cancer development and maintenance, although its function in lung cancer remains controversial. Here we demonstrate that TIP60 knockdown in non‐small cell lung cancer cell lines decreased tumor cell growth, migration, and invasion. Furthermore, analysis of a mouse lung cancer model with lung‐specific conditional Tip60 knockout revealed suppressed tumor formation relative to controls, but no apparent effects on normal lung homeostasis. RNA‐seq and ChIP‐seq analyses of inducible TIP60 knockdown H1975 cells relative to controls revealed transglutaminase enzyme (TGM5) as downstream of TIP60. Investigation of a connectivity map database identified several candidate compounds that decrease TIP60 mRNA, one that suppressed tumor growth in cell culture and in vivo. In addition, TH1834, a TIP60 acetyltransferase inhibitor, showed comparable antitumor effects in cell culture and in vivo. Taken together, suppression of TIP60 activity shows tumor‐specific efficacy against lung cancer, with no overt effect on normal tissues. Our work suggests that targeting TIP60 could be a promising approach to treating lung cancer.


| INTRODUC TI ON
Lung cancer is the leading cause of cancer-related deaths in the USA and worldwide. Despite the marked development of treatments such as targeted therapy or immunotherapy in the past two decades, more than 100,000 Americans died from lung cancer in 2021. 1  Conversely, TIP60 also functions in p53 activation, contributing to the induction of apoptosis [5][6][7] and is required for expression of KAI1, a tumor suppressor in prostate cancer. 8 Thus, TIP60 activity appears to be context dependent, and aberrant lysine acetyltransferase activity can either promote or suppress tumorigenesis in colon, breast, and prostate cancers. [8][9][10][11] Here, given that a function for TIP60 in lung cancer is also controversial, we investigated TIP60 function using in vitro cell culture and in vivo lung cancer models and searched for TIP60 effectors. We found that TIP60 serves as a coactivator to promote tumorigenesis in the context of lung cancer and that targeting TIP60 could serve as a treatment for lung cancer.

| MATERIAL S AND ME THODS
The materials and methods are described in Data S1.

| Low levels of TIP60 expression are required for tumorigenesis in lung cancer
First, we examined the TIP60 expression level in various human organs using GTEx datasets and found that TIP60 is ubiquitously expressed at various levels in multiple organ systems, including the lung ( Figure 1A). We then investigated TIP60 expression in various NSCLC lines: three (H1975, HCC827, and PC9) that harbor activating EGFR mutations, three (H358, H460, and A549) that harbor KRAS mutations, one each that harbors BRAF mutations (H1395), the EML4-ALK fusion gene (H3122), and a ROS1 fusion gene (HCC78), as well as BEAS-2B, an immortalized human bronchial epithelial cell line. Western blot analysis showed that all lines expressed four TIP60 splice variant isoforms at various TIP60 levels, although six out of nine NSCLC lines expressed lower levels of TIP60 than did BEAS-2B cells ( Figure 1B). To confirm these findings in clinical samples, we analyzed the data from TCGA lung adenocarcinoma dataset.
Comparative analysis revealed significantly lower TIP60 expression in lung adenocarcinoma relative to normal lung tissues ( Figure 1C), suggesting that TIP60 serves as a tumor suppressor in lung cancers.
To test this hypothesis, we selected H1975 and A549 (each harboring EGFR or KRAS mutations and both expressing lower TIP60) cells, and established the TIP60 overexpressing (OE) cells. Immunoblot analysis and qPCR confirmed TIP60 overexpression in H1975-OE cells and A549-OE cells ( Figure 2A, and Figure S1A); however, contrary to our hypothesis, TIP60 OE did not decrease cell growth of either line ( Figure 2B,C). Furthermore, TIP60 OE had no significant effect on migration and invasion in H1975 ( Figure 2D,F) and A549 cells ( Figure 2E,G). Different splice variant isoforms also had no significant effects on tumor growth (Figure S1B-F), suggesting that TIP60 does not function as a tumor suppressor in lung cancer.
Next, to investigate the effects of TIP60 silencing in cell culture, we tried to generate TIP60 knockout cells in H1975 using CRISPR-Cas9 but obtained no homozygous knockout cells (data not shown).
Thus we generated H1975 or A549 cells that harbored DOXinducible small hairpin RNA (shRNA) targeting TIP60. Doxycycline treatment of both lines specifically suppressed TIP60 expression at both mRNA ( Figure S2A,B) and protein levels ( Figure 3A,B) in two different shRNA clones of each (shTIP60-1 and shTIP60-2). We then comparable antitumor effects in cell culture and in vivo. Taken together, suppression of TIP60 activity shows tumor-specific efficacy against lung cancer, with no overt effect on normal tissues. Our work suggests that targeting TIP60 could be a promising approach to treating lung cancer.

K E Y W O R D S
artemisinin, KAT5, lung cancer, TGM5, TIP60 observed that, following DOX treatment and consequent TIP60 suppression, H1975 and A549 cells (each harboring shTIP60-2) significantly decreased cell growth relative to controls not treated with DOX ( Figure 3C,D). To assess migration activity in these lines, we performed a wound healing assay and found that TIP60 knockdown significantly decreased H1975 cell migration by day 1 ( Figure 3E) and A549 cell migration by day 3 ( Figure 3F). Furthermore, TIP60 knockdown significantly suppressed invasive activities of H1975 and A549 cells relative to controls not treated with DOX ( Figure 3G,H). Analysis in both lines using corresponding shTIP60-1 clones showed comparable cell growth, migration, and invasion activities ( Figure S2C-E).
To confirm that enzymatic activity of TIP60 is required for tumorigenesis, constructs of wild-type (WT) TIP60, TIP60-G380A (inactive mutation, mut) 12,13 or an empty vector were transiently transfected into TIP60 knockdown cells (H1975 shTIP60-2 under DOX treatment). Immunoblot analysis confirmed continuous suppression of TIP60 with DOX treatment in empty transfected cells, and TIP60 overexpression in TIP60-WT, or TIP60-mut transfected cells ( Figure S3A). Wild-type TIP60 increased the acetylation of Histone H4 ( Figure S3A), resulting in a significant increase in tumor cell growth, migration, and invasive activities, although TIP60 carrying the inactive mutation had no effect on acetyltransferase activity nor tumor growth ( Figure S3A-D). Interestingly, the knockdown of TIP60 expression in BEAS-2B cells showed no effect on cell growth and migration activities ( Figure S4A-C). Taken together, these results indicated that lung cancers showed relatively low TIP60 expression, which is essential for their survival, and that further suppressing TIP60 acetyltransferase activity antagonizes their tumorigenicity.

| Tip60 knockout inhibits tumorigenesis in mouse lung cancer
To assess the effects of TIP60 silencing in vivo, we generated  Figure 4E). These results indicate that TIP60 expression is required for lung tumorigenesis.

| Identification of candidate TIP60 targets by RNA-seq and ChIP-seq
Next, to identify TIP60 effectors, we performed RNA-seq and ChIPseq in shTIP60-1, shTIP60-2, and control H1975 cells. Analysis for DEGs revealed a large number of both downregulated and upregulated genes in H1975-shTIP60 cells relative to control cells, with more genes being downregulated ( Figure 5A,B). Furthermore, hierarchical clustering analysis of DEGs showed consistent expression changes in H1975-shTIP60-1 and shTIP60-2 relative to control cells ( Figure 5C). TIP60 reportedly acetylates multiple lysine residues on histone H4 4 ; thus we used an antipan-acetyl H4 antibody for ChIPseq. As shown in Figure 5D and Figure S6A, we identified 13 overlapping genes whose expression and histone H4 acetylation signal were both reduced by TIP60 suppression. Among them, we first focused on TGM2, a member of the transglutaminase family, which is reportedly associated with tumor growth or patient poor prognosis in colorectal carcinoma, glioblastoma, and pancreatic cancer [17][18][19] ; however, TGM2 knockdown had no effect on tumor growth in H1975 cells (data not shown). Thus we next focused on TGM5, which is also a member of the transglutaminase family. H4 acetylation levels at the TGM5 gene significantly decreased in both H1975-shTIP60-1 and -2 cells relative to controls ( Figure 5E), suggesting that TGM5 is a target of TIP60.
We next examined TCGA database to investigate Kaplan-Meier survival analysis of lung adenocarcinoma patients. The median overall survival of the patients in the high TGM5 score group was significantly shorter than that of the patients in the low TGM5 group

| Targeting TIP60 suppresses tumor progression in lung cancer
To identify compounds that might inhibit TIP60 expression, we used a connectivity map 20 and detected five high-scoring compounds ( Figure 6A). We investigated TIP60 inhibiting efficacy among these compounds, and found that artemether inhibited TIP60 expression relative to other compounds in both H1975 and A549 cells ( Figure S7A). Artemether is a derivative of artemisinin, which is a natural product derived from the Chinese herb Artemisia annua L. and widely used as an antimalarial drug. 21 Artemisinin downregulated TIP60 expression at lower concentrations than artemether in H1975 and A549 cells ( Figure 6B and Figure S7B); thus we selected artemisinin for further experiments. Dosedependent downregulation of TIP60 expression was observed in H1975 and A549 cells but less potent TIP60 downregulation in BEAS-2B cells ( Figure 6B). Interestingly, BEAS-2B cells showed significantly a higher IC 50 for artemisinin than did H1975 or A549 F I G U R E 4 Tip60 knockout lung cancer model mice show no tumor formation in the lung. (A) PCR of genomic DNA to confirm the genotypes of homozygous Tip60 knockout (F/F), heterozygous Tip60 knockout (F/wt), or wild-type (wt/wt) mice, as well as the EGFR transgene: I, CCSP-rtTA/Cre/Tip60 F/wt , II, EGFR TL /CCSP-rtTA/Cre/Tip60 wt/wt , III, EGFR TL /CCSP-rtTA/Cre/Tip60 F/wt , and IV, EGFR TL /CCSP-rtTA/ Cre/Tip60 F/F . (B, C) Appearance and weight of lungs in each mouse group: I (n = 14), II (n = 13), III (n = 24), IV (n = 14). Data are the mean ± SD. The p-value was calculated using one-way ANOVA followed by the Tukey-Kramer multiple-comparison test. **p < 0.005. ns; not significant. (D, E) Hematoxylin and eosin staining (D) and magnetic resonance imaging (E) in mouse groups indicated above. Note that no tumors are seen in Tip60 knockout mice. Images are representative, and white arrows indicate tumors. Scale bars: 200 μm. cells ( Figure 6C). Accordingly, the MTS assay revealed significant inhibition of H1975 and A549 cell viability by artemisinin treatment compared with those of BEAS-2B cells ( Figure 6D). Furthermore, artemisinin treatment significantly inhibited cell growth, migration, and invasive activities relative to cells treated with DMSO in H1975 and A549 cells ( Figure S7C-H). Next, to determine whether artemisinin treatment induces cell death by suppressing TIP60, we treated H1975-OE, A549-OE, or corresponding control cells with artemisinin and assayed Caspase-3/7 activity as an apoptotic marker. Artemisinin treatment decreased TIP60 expression in both control and OE cells, although TGM5 expression was inhibited only in controls ( Figure 6E). Caspase-3/7 activity was increased by artemisinin treatment in H1975 and A549 control cells ( Figure 6F), while upregulation of Caspase-3/7 activity in response to artemisinin treatment seen in control cells was significantly attenuated in H1975-OE and A549-OE cells ( Figure 6F). These results suggest that artemisinin treatment induces NSCLC cell apoptosis.
Next, we generated mouse xenograft models by injecting A549-TIP60 OE or control (Cntl) cells subcutaneously into nude mice. After tumors reached optimal volume (100-200 mm 3 ), we administered artemisinin at 200 mg/kg or vehicle orally once daily for 4 weeks.
Artemisinin treatment did not alter body weight in either mouse line ( Figure S8A). However, artemisinin treatment significantly inhibited tumor growth in mice bearing A549-Cntl xenografts relative to vehicle-treated mice, whereas the antitumor efficacy of artemisinin treatment was less potent in mice implanted with A549-OE cells ( Figure 6G,H).
To demonstrate that TIP60 acetyltransferase activity is required for tumorigenesis, we treated lung tumors with TH1834, a TIP60 acetyltransferase inhibitor, 22 in cell culture and in vivo. TH1834 treatment suppressed histone H4 acetylation and TGM5 expression in a dose-dependent manner in H1975 and A549 cells ( Figure 7A). We next treated H1975 and A549 cells with TH1834 and observed a significant inhibition of cell growth, migration, and invasive activities in both cell lines ( Figure 7B-F). Last, to investigate the effects of TH1834 in vivo, we used xenograft mouse models injected with A549 cells. After tumors reached optimal volume (100-200 mm 3 ), we administered TH1834 at 10 mg/kg or vehicle intraperitoneally five times per week for 3 weeks. 23 Body weight remained unchanged by TH1834 treatment ( Figure S8B). TH1834 treatment significantly inhibited tumor growth in mice bearing A549 tumors relative to vehicle-treated mice ( Figure 7G,H). Taken together, these findings suggest that targeting TIP60 could have an antitumor effect in the context of lung cancer.

| DISCUSS ION
In this study, we showed that TIP60 knockdown in an in vivo model of lung cancer inhibits lung tumor formation and progression. We also reveal that TGM5 is downstream of TIP60 and contributes to tumor progression in lung cancer. Furthermore, artemisinin or TH1834 treatment suppressed tumor growth in cell culture and in vivo, suggesting that targeting TIP60 might be a novel treatment for lung cancer (Figure 8).  24 TIP60 has a sequence distinct from that of GCN5 and acetylates different histones, 26 suggesting that TIP60 rather than GCN5 is crucial for lung tumorigenesis. Here, we showed that TIP60 was expressed at low levels in multiple lung cancer lines and in clinical lung tumor tissues relative to expression seen in normal tissues, initially suggesting that TIP60 serves as a tumor suppressor. However, TIP60 overexpression neither inhibited tumor progression nor induced tumor cell apoptosis.
In fact, TIP60 knockdown in H1975 and A549 cells inhibited cell growth, migration, and invasion activities, which is consistent with a previous report. 27 In contrast, another paper reported the antitumor activity of TIP60 using lung cancer cells. 28  Mice were monitored for changes in tumor volume. Data are presented as the mean percentage change in tumor volume ± SEM. The p-value was calculated using the unpaired two-tailed Welch's t-test. *p < 0.05 and **p < 0.005. ns; not significant. findings are consistent with previous studies suggesting that some cancer cells require low TIP60 levels for survival and the observation that eliminating already low TIP60 protein levels induces cancer cell death. 27,29 Interestingly, homozygous Tip60 deletion had no overt effect on normal lung tissues, although Tip60 loss is lethal for embryogenesis and hematopoietic stem cell maintenance. 15,30 These results suggest that TIP60 function is context dependent and critical for lung cancer cells but not for normal lung cells. Furthermore, despite evidence that TIP60 serves as a coactivator, TIP60 overexpression did not promote tumor progression.
Assuming that the minimal expression required for TIP60 activity is set at a lower point in lung cancer and has already reached a plateau, TIP60 overexpression might have no effect on tumor progression. Indeed, 50% loss of TIP60 (via heterozygous knockout) still exceeded the threshold level for TIP60 activity, resulting in tumorigenesis, whereas loss of all TIP60 expression (via homozygous knockout) completely inhibited tumor formation in mouse lungs.
It has been previously reported that reducing PU.1 expression to 20% but not 50% of normal levels promoted the development of acute myeloid leukemia, 31 indicating graded control of tumor formation by specific molecules. Of note, the relative difference in TIP60 expression levels between normal (higher) and tumor (lower) cells indicates that TIP60 suppression could have tumorspecific efficacy against lung cancer.
Our ChIP-seq and RNA-seq analyses revealed TGM5 to be downstream of TIP60, and a potential contributor to tumor progression and migration capacity. In addition, involucrin, which is cross-linked to membrane proteins by transglutaminase, and TGM2 are also among the 13 genes found to be downstream of TIP60, suggesting that transglutaminase plays specific roles in TIP60 regulatory activities. Transglutaminase family members are cross-linking enzymes that catalyze the formation of isopeptide bonds between the γ-carboxamide group of protein-bound glutamine and the ε-amino group of lysine residues, 32 and they function in cell adhesion, differentiation, and signal transduction. 33,34 However, TGM5 function in cancer remains unclear, whereas TGM2 is known to promote tumor cell differentiation, mobility, invasion, and survival. 33 To efficiently inhibit TIP60, we used a connectivity map database and selected artemisinin. We report that artemisinin treatment inhibited TIP60 expression and tumor progression. TIP60 regulates transcription of various genes, including NF-κB, MYC, and CCND1, [39][40][41] all also blocked by artemisinin and associated with antitumor activities of artemisinin, [42][43][44] suggesting that artemisinin exerts multiple anticancer effects via TIP60 regulation. However, artemisinin is not an ideal and specific compound for clinical suppression of TIP60 in part because high concentrations are required to inhibit TIP60 expression.
To improve its bioavailability and efficacy, several derivatives have been synthesized, among them dihydroartemisinin and artesunate. In early phase clinical trials, combination therapies, including artemisinin derivatives, with chemotherapy showed safety and high efficacy, 45,46 although the data were limited and larger scale phase clinical trials are needed. Artemisinin may also have antitumor activity independent of TIP60 inhibition. Accordingly, we utilized TH1834, a TIP60 acetyltransferase inhibitor, to treat lung tumors in cell culture and in vivo.
Tumor growth was suppressed, although a high concentration was still required for treatment. Therefore, a novel drug that specifically targets TIP60 is urgently needed. Recently, the degradation of targeted proteins using PROTACs has emerged as a promising therapeutic modality. PROTACs consist of three parts-a ligand of the protein of interest (POI), a ligand of an E3 ubiquitin ligase, and a linker-induce ubiquitylation and subsequent proteasomal degradation of the POI. 47 As PROTACs can target epigenetic proteins 48 or undruggable targets such as DNA-binding proteins (e.g., transcription factors), 49 the generation of a PROTAC targeting TIP60 could be an attractive approach to lung cancer treatment.
In summary, we show here that TIP60 is required for the malignant transformation of pulmonary epithelial cells. Understanding this mechanism is significant, as it could lead to novel and muchneeded therapy for lung cancer.

ACK N OWLED G M ENTS
We thank all members of the Kobayashi laboratory for helpful discussions.

FU N D I N G I N FO R M ATI O N
This work was supported by NIH CA240257 (HW, SSK), CA197697 (DGT), and CA218707 (DBC).

DATA AVA I L A B I L I T Y S TAT E M E N T
The RNA-seq and ChIP-seq data are available from the NCBI GEO database (GSE207202 and GSE207201). All other data are available in the main text or the supplementary materials.