Lysyl hydroxylases are transcription targets for GATA3 driving lung cancer cell metastasis

Metastasis associates with late stages of lung cancer progression and remains the main cause of patient death due to the lack of clinically effective therapeutics. Here we report that the transcription factor GATA3 and its co-factor FOG2 commonly promote the expression of the lysyl hydroxylase (LH) family members, including LH2 and LH3, which in turn drive lung adenocarcinoma cell migration, invasion, and metastasis. We show evidence that both LH2 and LH3 are direct transcription targets for GATA3. Knockdown of either LH2 or LH3 suppresses migration and invasion; on the contrary, forced expression of LH2 or LH3 promotes growth and migration, suggesting that the two LHs exert redundant oncogenic functions. Importantly, re-expression of LH2 is sufficient to restore the metastatic capacity of GATA3-depleted cells, suggesting a role for LHs as the downstream mediators of GATA3. Collectively, our data reveal a pro-metastatic GATA3-LHs axis for lung cancer, supporting the notion that targeting LHs may be useful for treating lung cancer.

cells, where it drives metastasis 19 . These results lead us to postulate that common transcription targets of FOG2 and GATA3 may be the drivers of metastasis and serve as putative therapeutic targets for treating metastatic lung cancer.
To identify the common targets of FOG2 and GATA3, we performed RNA-sequencing for metastatic lung adenocarcinoma cells expressing shRNAs against FOG2 or GATA3 19,20 . Among the common FOG2-and GATA3-regulated genes, we found that GATA3 promotes the expression of LH2 and LH3 by directly binding to their promoter elements. In vitro, forced expression of either LH2 or LH3 promotes lung adenocarcinoma cell growth and migration, and knockdown of LH2 or LH3 suppresses migration and invasion. In vivo, re-expression of LH2 in GATA3-depleted cells facilitates their xenograft tumor growth and metastatic capacity. Taken together, our findings suggest that LHs are pro-metastatic mediators of FOG2 and GATA3 and may be useful candidate therapeutic targets for treating lung cancer.

Materials and Methods
Cell culture and reagents. Lung adenocarcinoma cancer cell lines, including 393 P, 344SQ, H1299, and HCC827 cells, were cultured in a humidified atmosphere with 5% CO2 at 37 °C in RPMI 1640 medium supplemented with 10% FBS. Anti-flag antibody (clone M2) was purchased from Sigma. Rabbit anti-GATA3 polyclonal antibody (sc-9009) and normal rabbit IgG were purchased from Santa Cruz. Rabbit anti-Tubulin (2125) antibody was purchased from Cell Signal Technology. Human LH2 and LH3 expression plasmids were gifts from Jonathan Kurie MD (the University of Texas MD Anderson Cancer Center). Human and mouse PLOD2 siRNA were purchased from OriGene and Santa Cruz, respectively. Mouse PLOD3 siRNAs were purchased from Santa Cruz. The ChIP essay kit was purchased from ActiveMotif (#53009).
Cell lysate preparation and Western blotting. Cells were washed by PBS and lysed in a RIPA lysis buffer (Santa Cruz Biotechnologies). Total protein lysates were quantified for protein concentrations with a BCA protein assay kit (Pierce Biotechnology). Protein samples were separated on denaturing SDS-polyacrylamide gel and transferred to a nitrocellulose membranes (Amersham Hybond ECL; GE Healthcare), followed by incubating with appropriate primary antibodies overnight at 4 °C. After being washed with TBST for 30 minutes at room temperature, the membrane was further incubated with HRP-conjugated secondary antibodies for 1 hour, followed by 30 minutes of washing with TBST. Protein bands were visualized with supersignal ECL substrates (Pierce Biotechnology). Uncut western blot gels are shown in Supplemental Fig. 1.
Cell transfection. Transient cell transfection were performed by using lipofectamine 2000 for DNA plasmids or lipofectamine RNAiMAX for siRNAs as described by the manufacturer (Invitrogen). To stably express LH2, the 393 P and GATA3 shRNA-expressing 344SQ lung cancer cells 20 were transfected with a pEF-bsr-FLAG-LH2 expression plasmid. 48 hours after transfection, cells were prepared into single cell suspension and cultured in the presence of blasticidin (10 μg/ml) for 1 week. To stably express LH3, the 393 P and HCC827 lung cancer cells were transfected with a pLVX-puro2-FLAG-LH3 expression plasmid. 48 hours after transfection, cells were prepared into single cell suspension and cultured in the presence of puromycin (1 μg/ml) for 2 week.
Chromatin immunoprecipitation assay. ChIP immunoprecipitation assay was performed using the ChIP assay kit according to the manual provided by the manufacturer (ActiveMotif). Briefly, H1299 cells were transiently transfected with GATA3 cDNA for 48 hours and were cross-linked with 1% formaldehyde and then incubated in lysis buffer on ice for 30 minutes. After being grinded several times at 4 °C, the nuclei were pelleted by centrifugation at 5,000 rpm for 10 min at 4 °C. The nuclei were then digested and the sheared chromatin was subjected to immunoprecipitations. For the immunoprecipitations, ChIP Grade anti-GATA3 antibody and normal rabbit anti-IgG antibody were used. Immunoprecipitated DNA was amplified by quantitative real-time PCR (qPCR) using an ABI 7500 fast instrument (Thermo Fisher Scientific Inc).
Luciferase-conjugated promoter reporter assay. Luciferase-conjugated human LH2 promoter reporter construct in the pGL3 basic vector (Promeaga) was generated by our previous studies 9 , and we cloned a ~1.5 k human LH3 promoter into the same vector using the following primers: F: 5′-GGGGTACCTCACTGCCTTTGCACGTGAG-3′, and 5′-CGGCTAGCACAGAAAGCTCCAGAT GCCG-3′. Reporter assays were performed as we described 9 . Briefly, cells were grown to 90% confluency in 24-well plates and transfected with the promoter reporter constructs or with the empty pGL3 vector (500 ng per well), and co-transfected with a pRL-TK (Promeaga, 10 ng per well) as internal control. 72 hours after the transfection, the luciferase reporter activities were measured with the Dual-Luciferase assay kit from Promeaga according to the manufacturer's datasheet.
Migration and invasion assays. Migration and invasion abilities of tumor cells were quantitated by using 24 well plate 8 μm pore size of noncoated (for migration assay) or matrigel-coated (for invasion assay) transwells as described by the manufacturer (BD Biosciences). Briefly, cells (1 × 10 5 per well) were seeded in the upper chambers in serum free RPMI 1640 cell culture medium (in triplicate), and RPMI 1640 medium containing 10% FBS was added to the bottom wells. After 18 hours of incubation, migrated or invaded cells were stained with Diff-Quik Stain Set (Siemens), photographed, and counted manually using Image-pro plus 4.5 (Media Cybernetics, Silver Spring, USA) software.

Mouse experiment.
To examine the in vivo effect of LH2 in the growth and metastasis, 344SQ cells expressing scrambled and GATA3 shRNAs were subcutaneously injected (5 × 10 5 cells per injection) into the flanks of 8 weeks old 129-elite mice (Charles River). Mice were watch three times a week and killed at 4 weeks after injection. No suspicious tumor growth, e.g. tumor regression, was observed during the experiment. At necropsy, both subcutaneous xenograft tumors and the lungs were dissected and fixed in formalin. All mouse experiments and protocols were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC), and the methods were carried out in accordance with the institutional guidelines and regulations.
RNA extraction and Quantitative RT-PCR. Cells were washed with ice-cold PBS, and subjected to RNA extraction using 700 ul of TRIzol (Invitrogen). mRNA was reverse transcribed using SuperScript IV (Invitrogen). For qPCR reactions, cDNA products were amplified using SYBR Green PCR Master Mix and analyzed using the 7500 fast Real-Time PCR System (both from the Applied Biosystems). mRNA expression levels were normalized to that of the ribosomal protein L32 mRNA by standard curve method.
Statistics. All statistical comparisons between 2 groups were performed using the 2-tailed Welch's t test.
One-way ANOVA was performed to compare multiple experimental groups. P values that are less than 0.05 are considered statistically significant. Data availability. RNA sequencing data and relevant data are available from the authors upon request.

Results
Our previous studies have shown that knockdown of GATA3 and its co-factor FOG2 suppresses spontaneous metastasis of 344SQ lung adenocarcinoma cells in syngeneic models of metastasis 19,20 . To determine whether FOG2 and GATA3 commonly regulate transcriptome changes to drive metastasis, we performed RNA-sequencing for 344SQ cells that express control scrambled shRNAs or shRNAs against FOG2 or GATA3. Consistent with our previous findings, knockdown of GATA3 significantly reversed EMT-related gene expression changes, as determined by increased expression of epithelial genes and decreased expression of mesenchymal genes (Supplemental Fig. 2A). On the contrary, knockdown of FOG2 had weaker or even opposite effects on their expression (Supplemental Fig. 2A), suggesting that the changes of these EMT-related genes may not be required for metastasis driven by the FOG2/GATA3 complex.
Moreover, our sequencing results revealed that the mRNA expression levels of more than 700 genes were commonly changed upon the knockdown of FOG2 and GATA3 (Supplemental Fig. 2B). Among the common FOG2-and GATA3-regulated genes, LH2 and LH3 were the most attractive for us, because recent results from us and others have shown that LH2 exerts a pro-metastatic function and may have a therapeutic value [7][8][9][10] . Sequence analysis showed that both LH2 and LH3 gene promoters contained putative binding sites for GATA3 (Fig. 1A,B), suggesting that they may be direct transcription targets for GATA3. In support of this idea, chromatin immunoprecipitation (ChIP) assays showed that GATA3 bound to multiple candidate GATA3-binding consensus sequences on both LH2 and LH3 promoters (Fig. 1A,B), and knocking down GATA3 inhibited the expression of both LHs (Fig. 1C) and inhibited their promoters' transcription activities (Fig. 1D). As additional controls, we performed ChIP assays for several other randomly selected GATA3-regulated genes, including SEMA4A, PDE4B, PIK3CD, and E030019813RIK. The results showed that GATA3 also bound to the promoter of SEMA4A (Fig. 1E), but not the other three genes (Fig. 1F) direct and indirect mechanisms. Therefore, it is likely that GATA3 simultaneously regulates multiple transcription targets, which in turn collectively mediate its biological functions.
To determine the role of LH2 and LH3, we knocked down or expressed LH2 and LH3 in multiple mouse and human lung cancer cell lines. We found that knocking down either LH2 or LH3 ( Fig. 2A,B) inhibited the invasiveness (Fig. 2C,D) and growth (Fig. 2E) of 344SQ cells. Similarly, knockdown of LH2 in HCC827 cells (Fig. 2F) suppressed their growth (Fig. 2G), migration (Fig. 2H,I), and invasion (Fig. 2J,K). On the contrary, forced expression of either LH2 or LH3 (Fig. 3A) could promote the soft agar colony formation ability (Fig. 3B), migration (Fig. 3C), and invasion of 393 P cells (Fig. 3D). Forced expression of LH2 or LH3 also promoted the migration of other lung tumor cells, including 344SQ (Fig. 3E,F), and HCC827 cells (Fig. 3G,H), precluding any potential specific effects of LH2 or LH3 expression in a single cell line. Consistent with these findings, knocking down GATA3 not only inhibited LH2 and LH3 expression (Fig. 1D), but also suppressed 393 P cell growth (Fig. 3I), migration (Fig. 3J), and invasion (Fig. 3K).These results suggest that LH2 and LH3 may be functionally redundant as the downstream mediators of GATA3, though they may differ in terms of extents in particular assays (Fig. 3B-H). More importantly, stable expression of LH2 alone was sufficient to promote xenograft tumor formation ability and to restore the spontaneous metastatic capacity of GATA3-depleted 344SQ cells (Fig. 4A,B and Table 1), suggesting that the loss of LH2 expression in GATA3-depleted cells ( Supplemental Figs 1 and 2) was critical for suppressing tumor growth and metastasis. Furthermore, mining the public Oncomine lung cancer datasets revealed that the expression levels of both LH2 and LH3 were upregulated in lung adenocarcinomas compared to normal lung tissues (Table 2), supporting the notion that these LHs may be useful targets for developing strategies for treating lung cancer.

Discussion
There are at least three lysyl hydroxylases (LH1-3) in mammalian cells, and it is known that they have differential enzymatic activities. For instance, although all three LHs catalyze the hydroxylation of lysine residues on collagen, LH2 can act as a specific telopeptide hydroxylase 21 . In addition, LH3 is not only a lysyl hydroxylase but also a glycosyltransferase under certain circumstances 22,23 . Interestingly, despite of these differences, our results show that the manipulation of either LH2 or LH3 has a significant impact on invasion and metastasis (Figs 2 and 3). It is unclear whether LH2 and LH3 exert such redundant functions through differential mechanisms. Therefore, it will be interesting for future studies to determine whether LH2 and LH3 exert such similar functions through their common lysyl hydroxylase activity, or through their distinct enzymatic activities as above-mentioned. Notably, recent studies have shown that both LH2 and LH3 have novel roles as secreted enzymes to remodel extracellular matrix 24,25 , raising another possibility that the two LHs may exert multiple functions associated with their intracellular or extracellular activities. Moreover, it has been shown that different LHs may have distinct substrate specificity and catalyze the hydroxylation of distinct members of collagens 26 , leaving it open to test whether the combined activities of LH2 and LH3 drive metastasis through the modification of multiple collagen isoforms rather than a specific one.
As a reversible cellular process, EMT has been implicated in metastasis in a variety of cancer types, including lung cancer, and many EMT-regulating factors (e.g. transforming growth factor β1, Snail, Slug, Twist, and ZEB factors) are involved in the regulation of metastasis [27][28][29][30][31][32][33][34][35][36][37] . On the other hand, EMT-independent metastasis has also been reported for breast and pancreatic cancers 38,39 . In support of the role of EMT in lung cancer metastasis, our previous studies have shown that GATA3 drives lung adenocarcinoma cell EMT, invasion, and metastasis through the transcriptional repression of miR-200, an epithelial small non-coding RNA that inhibits EMT by targeting ZEB factors 20 . From such an aspect, our findings that FOG2 and GATA3 differentially regulate EMT-related gene expressions suggest that both EMT-dependent and -independent pathways may collectively contribute to lung cancer metastasis, and GATA3 may act as a master regulator of both EMT-dependent and -independent metastasis and serve as a convergence point for the crosstalks between EMT-dependent andindependent pathways.   Table 2. Lung tumor/normal lung fold changes of LH2 and LH3 mRNAs in the Oncomine datasets.