The Twist Box Domain Is Required for Twist1-induced Prostate Cancer Metastasis

This article is featured in Highlights of This Issue, p. 1293

Prostate cancer is the most common cancer diagnosed in men in the United States and is responsible for the second most cancer deaths in men (1). Patterns of disease failure in prostate cancer suggest understanding the determinants that confer progression of localized presentations to metastatic disease will result in the largest therapeutic gains (2).

One mechanism by which cancer cells may acquire the characteristics necessary for metastasis is the epithelial–mesenchymal transition (EMT). EMT is a transcriptional program, crucial in early embryonic development that is co-opted by some cancer cells to facilitate aggressive and metastatic behavior (3). Twist1 is a basic helix-loop-helix (bHLH) multidomain transcription factor which directly mediates EMT by transcriptional activation and repression of E-box-regulated target genes (4, 5). A role for TWIST in prostate cancer pathogenesis has been suggested (6, 7), but the role of EMT and Twist1 in prostate cancer disease progression and metastasis is just now being explored (8, 9). The critical domains of Twist1 and the crucial Twist1 downstream transcriptional targets required for increased tumorigenicity and aggressive metastatic phenotypes in prostate cancer are unknown. The carboxyl-terminal Twist box is a highly conserved domain among Twist1 orthologues for which little functional information in the context of cancer phenotypes is known (5). A greater understanding of the structure–function relationships and downstream targets of Twist1 may allow for an increased appreciation of the mechanisms responsible for Twist1-induced metastasis and may facilitate more precise inhibitory strategies of Twist1 as a therapeutic maneuver in cancer.

Here, we used a single amino acid substitution mutation, Twist1 codon 191 phenylalanine-to-glycine (F191G), to study the role of the Twist box for Twist1-induced aggressive cellular and metastatic phenotypes in prostate cancer cells. Isogenic androgen-dependent, Myc-CaP (10), and androgen-independent, PC3, cell lines overexpressing Twist1 or the Twist box mutant showed specific requirements for the Twist box during Twist1-induced metastasis of prostate cancer cells. Gene expression profiling revealed transcriptional programs directed by the Twist box that were associated with cancer metastasis. Finally, we show that Twist1 directly regulates one such target, Hoxa9, which is partially required for Twist1-induced prostate cancer prometastatic phenotypes.

pBABE-Twist1-puro or –hygro (11) was used to construct the Twist1-F191G mutant using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and confirmed by sequencing. Antibodies used were: Twist (Twist2C1a; sc-81417, Santa Cruz Biotechnology), E-cadherin (ab53033, Abcam), vimentin (ab92547), ZO-1 (5406, Cell Signaling Technology), β-actin (A5316, Santa Cruz Biotechnology), c-Myc (N-term; 1472-1, Epitomics), horseradish peroxidise-conjugated secondary antibodies (Invitrogen), and Alexa flour 488 conjugated secondary antibodies (Invitrogen). Hoxa9 shRNA retroviral constructs were purchased and used as directed by Origene (cat #TG500979).

PC3 and 22RV1 were obtained from American Type Culture Collection. Myc-CaP was a kind gift from Dr. John Isaacs (Johns Hopkins University, Baltimore, MD; ref. 10). Growth media: Myc-CaP, Dulbecco's modified Eagle medium (Invitrogen); PC3, Hams F12K (Invitrogen); and 22RV1, RPMI-1640 (Invitrogen). Cell line identity confirmed by short tandem repeat profiling and mycoplasma tested. All media were supplemented with 10% FBS and penicillin (100 U/mL), streptomycin (0.1 mg/mL). Cells were maintained at 37°C in a humidified incubator with 5% CO₂ in air.

Retroviral production used ecotropic and amphotropic Phoenix packaging lines. Myc-CaP cells were transduced with pGFP-V-RS-based shRNA contructs from Origene as described above or with scrambled control vector for two successive times over a 36-hour period followed by selection with 1 mg/mL puromycin and passaged once 80% confluent.

Subconfluent cells were transfected using Lipofectamine 2000 (Invitrogen) with 200 ng of firefly luciferase reporter gene construct (100 ng was used for SNAI2 reporter assays), 100 ng of the pRL-SV40 Renilla luciferase construct, and 500 ng of the Twist1 or Twist1-F191G–mutant expression construct. Cell extracts were prepared 36 hours after transfection in passive lysis buffer, and the reporter activity was measured using the Dual-Luciferase Reporter Assay System (Promega).

Two-dimensional migration assay was conducted using a scratch/wound model. Cells were grown in 6-well plates for 24 hours to confluence. PC3 cells were treated with 500 pmol/L TGF-β at the time of wounding. Multiple scratch wounds were created using a P-20 micropipette tip and cells fed with fresh complete media. Five representative fields of the wound were marked and images were taken at 0 and 24 hours after wounding. Relative wound closure is calculated from the remaining wound area normalized to the initial wound area using ImageJ software (NIH Image).

Fourier transform traction microscopy (FTTM) was used to measure the contractile stress arising at the interface between each adherent cell and its substrate as described (12). Briefly, cells were plated sparsely on elastic collagen type I coated gel blocks. Images of fluorescent microbeads (0.2 μm in diameter, Molecular Probes) embedded near the gel apical surface was taken at different times with cell-free reference (traction-free) images. The displacement field between a pair of images was then obtained by identifying the coordinates of the peak of the cross-correlation function (13, 14). From the displacement field and known elastic properties of the gel (Young's modulus of 1 kPa with a Poisson's ratio of 0.48), the cell traction field was computed. The computed traction field was used to obtain net contractile moment, which is a scalar measure of the cell's contractile strength, expressed in pico-Newton meters (pNm).

Magnetic twisting cytometry (MTC) was used to measure material properties of the cytoskeleton as described (15, 16). In brief, cells were plated at 150,000 cells/cm² on coated collagen type I plastic wells (96-well Removawell, Immulon II: Dynetech) at 500 ng/cm². After scratching with a 200-μL pipette tip and the indicated time, ferrimagnetic microbeads were functionalized to the cytoskeleton, and both stiffness, g′ and loss modulus g″, were measured over a physiologic range of frequency (f) expressed in Pascal per nm (Pa/nm).

The invasion potential was assessed using Chemicon cell invasion assay kit (Millipore) as directed by the manufacturer. Of note, 8 μmol/L Transwells with Matrigel were used for the assay. Serum-starved 0.5 to 1 × 10⁶ cells (12–16 hours) in 300 μL were seeded in upper chambers, whereas lower wells were filled with 500 μL of 10% FBS complete medium. Invading cells on the lower surface were fixed and stained. The stain is dissolved in 200 μL of 10% acetic acid and measured at 570 nm. Invasive potential is derived by normalizing with the readings from blank Transwell inserts.

Immunohistochemistry (IHC), immunofluorescence, and Western blotting were conducted as described previously (17).

Anoikis resistance was measured using a modified protocol (18). Cells were grown in normal attachment and ultra-low attachment (Corning) in 6-well plates. Twenty-four hours later, cells were blocked in 5% FBS and stained with Alexa Fluor 488 conjugated AnnexinV followed by propidium iodide staining (50 μg/mL; Invitrogen). Cells were enumerated on a BD FACS Caliber (BD Biosciences), and analysis was done using FlowJo analysis software. All conditions were n = 4 and two replicates per experiment.

Clonogenic survival was conducted as previously described (19). Soft agar clonogenic assays used 6-well plates precoated with 1 mL of basal 0.6% agarose in complete media and overlaid with 2 mL of cells (5 × 10³ cells/mL) mixed with 0.3% agarose in complete media and allowed to solidify. The wells were constantly fed with complete media to prevent drying of agarose, and then after 10 to 15 days of incubation, colonies were scored under phase contrast microscopy. All conditions were repeated at least twice with 3 wells per experiment.

All procedures were carried out in accordance with the Johns Hopkins Animal Care and Use Committee, maintained under pathogen-free conditions, and given food and water ad libitum. For the subcutaneous tumor graft assay, 100 μL of PBS and Matrigel (BD Biosciences) mixed 1:1 containing 0.5 to 2 × 10⁶ cells were injected subcutaneously into both the flanks of 8-week-old male FVB/N or athymic nude mice. Tumors measured 3 times weekly and volume calculated: length × width × height × π/6. Tumor growth delay is the difference between the quadrupling times of untreated versus treated tumors. For the experimental lung metastasis assay, 100 μL of PBS containing 5 × 10⁵ cells were injected into athymic nude mice via the tail vein. After 4 weeks, the mice were sacrificed, necropsies conducted to score surface lung tumors and extrathoracic metastases.

Microarrays were conducted using GeneChip WT cDNA Synthesis and amplification Kit and WT terminal labeling Kit (Affymetrix). The labeled ssDNA was hybridized to the GeneChip Mouse Gene 1.0 ST array (Affymetrix), washed with the Fluidics station 450, and array scanning was conducted as previously described. Arrays were normalized using the Robust Multichip Average in the oligo Bioconductor package at the transcript level. Genes and gene sets with Benjamini–Hochberg P < 0.05 were considered statistically significant. Gene set enrichment analysis (GSEA) was conducted using the C2 Curated Gene Sets collection from the Molecular Signature Database 3.0 and statistical comparisons by Fisher Exact test. More detailed description of the analysis and R code used for this analysis are included as Supplementary Materials and Methods.

Chromatin immunoprecipitation (ChIP) was conducted using a SimpleChIP Enzymatic IP Kit (Cell Signaling Technology). See Supplementary Materials and Methods for details.

The iTaq Universal SybrGreen Master Mix (BioRad) was used according to the manufacturer's instructions. Human normal prostate and prostate cancer qPCR tissue arrays and TWIST1 qPCR oligos were purchased from OriGene. All relevant clinical information can be found in http://www.origene.com/qPCR/Tissue-qPCR-Arrays.aspx.

Statistical analysis was carried out using GraphPad Prism v5.04 (GraphPad Software). Paired comparisons were tested using the Mann–Whitney test or Fisher exact test. Throughout this study: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

TWIST1 expression in prostate cancers was analyzed from 14 independent microarray datasets constituting 1,013 prostate samples (20–32) using Oncomine. Ten of the 14 datasets and the aggregate analysis showed TWIST1 overexpression in prostate cancers (Fig. 1A, P = 0.002 for aggregate). Further analysis of one of these microarray studies (20) showed that TWIST1 overexpression correlated with metastatic disease (Fig. 1B; P < 0.000001).

This microarray data were validated using quantitative PCR (qPCR) for TWIST1 on human prostate cancer samples. Cancer samples (n = 107) screened by qPCR confirmed that TWIST1 was overexpressed in prostate cancer (40/107 or 37% showed ≥3-fold upregulation, 18/107 or 17% ≥10-fold overexpression, and some cases ≥50-fold overexpression; Fig. 1C; P < 0.0001). Similar to the microarray data, TWIST1 overexpression was directly correlated with prostate cancer aggressiveness as determined by Gleason score (Fig. 1D; P < 0.0001). These data agreed with prior studies that showed TWIST1 overexpression in human prostate cancer and correlation with prostate cancer disease aggressiveness and metastasis (7, 33).

Twist1 has four domains: (i) a Twist1 domain that is highly conserved among human and mice; (ii) a glycine rich region; (iii) bHLH domain; and (iv) the Twist box (or WR domain). The Twist box is identical between mouse, human, frog, zebra fish, and jellyfish species and is located in the last 23 residues of the mouse polypeptide. The Twist box has been shown to be necessary and sufficient to transactivate E-box containing heterologous reporter constructs in vitro (34). We generated a site-specific Twist box mutant by mutating a critical Phe-191 to Gly, referred to as Twist1-F191G (Fig. 2A). This Twist1-F191G mutant had been shown previously to be deficient for transactivation of E-box containing reporter constructs in mesenchymal cells (34), but has not been examined in epithelial cancer cells. In Myc-CaP androgen-dependent prostate cancer cells, Twist1 overexpression significantly repressed CDH1 (Fig. 2B, left, P < 0.001) promoter activity (35) and increased SNAI2 (Fig. 2B, right, P < 0.001) promoter activity. Twist1-F191G was found to be defective for both repression and activation in these assays and significantly different from Twist1 (Fig. 2B; P < 0.05 for both). There was some suggestion that Twist1-F191G was more defective for activation than repression, as Twist1-F191G was still able to repress the CDH1 promoter to some extent but could not activate the SNAI2 promoter as compared with Vector control (Fig. 2B, left, P < 0.05 and right, P = 0.0952). Similarly, in HEK 293 cells, Twist1 repressed CDH1 promoter activity, whereas Twist1-F191G only partially repressed the CDH1 promoter activity compared with Twist1 wild-type (Supplementary Fig. S1A, both P < 0.001). Neither Twist1 nor Twist1-F191G seemed to alter expression from the SNAI2 promoter in HEK 293 cells, which is not surprising as this cell line is of likely mesenchymal origin (Supplementary Fig. S1B; all P>0.05). These reporter assay data were concordant with levels of the endogenous Cdh1 and Snai2 genes and respective gene products when Myc-CaP cells overexpressed Twist1 or Twist1-F191G stably (Fig. 2C and Supplementary Fig. S1C–E). Twist1-F191G bound the Cdh1 promoter as well as Twist1 wild-type in Myc-CaP cells according to ChIP-qPCR (Supplementary Fig. S1F). These results suggest that the Twist box domain is required for the full transcriptional activity of Twist1.

Metastasis is a complex series of discrete events that a neoplastic cell must traverse (36). These serial events include: loss of cell-to-cell adhesion, migration and invasion into the local extracellular matrix, intravasation into the vasculature, resistance to anoikis, extravasation into the parenchyma of distant tissues, and then colonization into a macroscopic metastatic tumor. To ascertain the role of the Twist box domain in a subset of these metastatic steps in vitro and in vivo, stable isogenic cell lines expressing Twist1 and Twist1-F191G in Myc-CaP and PC3 prostate cancer cells were established (Western blot analysis, Fig. 2C; and immunofluorescence, Fig. 2D, top row and Supplementary Fig. S2A and S2B). Consistent with an EMT marker profile, stable Twist1 overexpression led to downregulation of epithelial markers E-cadherin and ZO-1 in Myc-CaP cells and in PC3 cells and upregulation of the mesenchymal marker vimentin in both Myc-CaP and PC3 cells (Western blotting and immunofluorescence shown in Fig. 2C and D and qPCR shown in Supplementary Figs. S1C and S3A). The Twist box mutation, Twist1-F191G, resulted in a reduced ability to downregulate E-cadherin and ZO-1 and upregulate vimentin in Myc-CaP cells (Fig. 2C and D and Supplementary Figs. S1C and S3A). A similar, but less dramatic loss of an EMT marker profile was seen with PC3 cells stably overexpressing Twist1-F191G (Fig. 2C and D; Supplementary Fig. S2C and S2D show immunofluorescence quantification of PC3 cells; and Supplementary Fig. S3B and S3C show qPCR for CDH1 and VIM). The findings from these prostate cancer cell lines overexpressing Twist1 and Twist1-F191G suggest that the Twist1 box domain is required for Twist1 to induce a full EMT marker profile.

We next examined Twist1-induced cell migration and correlates of cell mechanics using the scratch/wound-healing model. For this study, we made a scratch into an ensemble of confluent Myc-CaP cells (Fig. 3A) and measured the spatiotemporal changes in forced motions of microbeads anchored to the cytoskeleton through integrin cell adhesion receptors (15, 16). Using MTC, we measured cytoskeletal stiffness (g′) and internal friction (g″) before, immediately after, and 24-hours after making a scratch. Over five decades of frequency, we found no differences in stiffness g′ and friction g″ between isogenic Myc-CaP cell ensembles (Twist1 vs. Vector) before or immediately after a scratch (Supplementary Fig. S4A and S4B). By 24 hours, however, Twist1-overexpressing cells infiltrated more into the site of the scratch wound than Vector control cells (Fig. 3A) and showed appreciably higher cytoskeletal stiffness (Fig. 3B and Supplementary Fig. S4c) and friction (Fig. 3C and Supplementary Fig. S4C). At 24-hour after making a scratch, Twist1-overexpressing cell ensembles exhibited a 1.6-fold higher stiffness than Vector control ensembles (Fig. 3D). Most striking was the greatest differences of cytoskeletal stiffness between Twist1 and Vector control cell ensembles were localized at the leading migratory front (Fig. 3A and E). Twist1-overexpressing cell ensembles had progressively decreasing cytoskeletal stiffness with increasing distance from the leading migratory edge (1st > 2nd > 3rd > 4th cell layers; Fig. 3A and E).

We then directly assessed the requirement of the Twist box domain for Twist1-induced cell invasive potential using the scratch/wound-healing assay in vitro. Importantly, Twist1 overexpression did not increase the proliferative potential of Myc-CaP or PC3 cells (Supplementary Fig. S5). Myc-CaP cells overexpressing Twist1 migrated 2.5-fold faster than Vector control Myc-CaP cells (Fig. 3F and G, P < 0.05). The Twist1-F191G–overexpressing cells were significantly less migratory than Twist1-overexpressing cells, but still migrated more than the Vector (1.5-fold; Fig. 3F and G, both comparisons P < 0.05). The same trends for cell migration were also observed in PC3 cells (Supplementary Fig. S6).

Cancer cell migration and invasion entail the ability of malignant cells to exercise contractile force upon their surroundings (37). Using traction microscopy (13, 14), we interrogated the force-generating capacity of single Myc-CaP and PC3 cells overexpressing Twist1 (Fig. 3H–J and Supplementary Fig. S7). Compared with Vector, the Twist1-overexpressing Myc-CaP and PC3 cells showed increased cell spreading area and net contractile moment, a scalar measure of the cell's contractile strength (Fig. 3I and J, P < 0.01 both measurements and Supplementary Fig. S7B and C, P < 0.001 both measurements). The Twist box mutant displayed less cell spreading and lower net contractile moment compared with Twist1 (Fig. 3I and J, P < 0.05 for both measurements). These single-cell biophysical data corroborated our results with bulk migration assays. Collectively, these data suggested that the Twist box domain was required for increasing cytoskeletal force generation in prostate cancer cells that was associated with the full migratory potential of Twist1.

Both Myc-CaP and PC3 cells overexpressing Twist1 showed an increased invasiveness compared with Vector control cells using a Matrigel-coated Transwell invasion assay (Fig. 4A and B, both P < 0.05). The Twist box mutant was completely defective for invasion in Myc-CaP cells compared with Twist1 wild-type (Fig. 4A, P < 0.05) and trended toward being less invasive in PC3 cells (Fig. 4B, P = 0.156). Similar to motility, the Twist box is at least partially required for Twist1-induced prostate cancer invasion.

The resistance to anoikis facilitates metastasis of cancer cells to distant organs. Both Twist1-overexpressing Myc-CaP and PC3 cells showed decreased apoptosis when grown in suspension compared with their isogenic Vector control cells (Fig. 4C–F, both cell lines P < 0.05). The Twist1-F191G mutant in Myc-CaP and PC3 cells were similar to their isogenic Vector control cells (Fig. 4C–F, both cells P>0.47) for anoikis resistance. We also observed that the Twist box was required to confer radioresistance to prostate cancer cells (Supplementary Fig. S8). Altogether, these data show that Twist1 overexpression can confer resistance to multiple cell death stimuli, and that the Twist box domain is required for these Twist1 activities in prostate cancer cells.

The in vitro anchorage-independent growth of Myc-CaP and PC3 cells stably overexpressing Twist1 and the Twist box mutant was conducted (Fig. 4G–J). Both Twist1-overexpressing Myc-CaP and PC3 cells showed increased frequency of colonies in soft agar compared with their isogenic Vector control cells (Fig. 4G–J, both cells P < 0.01). In addition, Myc-CaP cells overexpressing Twist1 had colonies of larger size (Fig. 4G). Myc-CaP and PC3 Twist box mutant cells had a similar frequency of colonies in soft agar compared with their isogenic Vector control cells (Fig. 4G–J, both cells P>0.20). These general results were repeated and confirmed in a third prostate cancer cell line, 22Rv1, stably overexpressing Twist1 and Twist1-F191G (Supplementary Fig. S9). These data further confirm the importance of the Twist box domain for aggressive in vitro prostate cancer cell behavior induced by Twist1.

Using a subcutaneous tumor graft assay, we did not observe Twist1 or Twist1-F191G overexpression increasing the in vivo primary tumorigenic potential or primary tumor growth of Myc-CaP, PC3, or 22Rv1 cells (Supplementary Fig. S10). The metastatic potential of Twist1 and Twist1-F191G–overexpressing Myc-CaP cells was assessed using the experimental lung metastasis assay. Twist1 overexpression significantly increased the ability of Myc-CaP cells to colonize the lungs and form macroscopic metastases in vivo (Fig. 5A and B; 10/18 mice with Twist1-overexpressing Myc-CaP cells vs. 2/17 mice with isogenic Vector control cells, P = 0.0116). The Twist box mutant overexpressing Myc-CaP cells lost some potential to form macroscopic lung metastases in vivo and had an intermediate phenotype to Vector and Twist1 Myc-CaP cells (Fig. 5B; 4/12 mice with Twist1-F191G–overexpressing Myc-CaP cells, P>0.2). The tumor cell morphology from Twist1 and Twist1-F191G–overexpressing cells was not different (Fig. 5C). Interestingly, mice injected tail vein with Twist1-overexpressing Myc-CaP cells showed extrathoracic metastases to distant subcutaneous tissues, abdominal organs, and distant lymph nodes (Fig. 5D–F). Cells injected in the venous circulation seed the lungs and therefore must undergo the full metastatic pathway to produce extrathoracic metastases. Twist1 overexpression significantly increased the frequency of mice with extrathoracic metastases (Fig. 5E; 11/18 mice with Twist1-overexpressing Myc-CaP cells compared with 1/17 mice with isogenic Vector control cells, P = 0.0009). The Twist box was required for Myc-CaP cells to undergo the full metastatic pathway and give rise to extrathoracic metastases (Fig. 5E; 2/12 mice injected with Twist1-F191G–overexpressing Myc-CaP cells, P = 0.0256 compared with Twist1; and P = 0.553 compared with Vector control cells). The Myc-CaP identity of lung tumors and extrathoracic metastases was confirmed by Myc IHC (Fig. 5G). These results show that the Twist box domain is required for Twist1-induced metastasis of prostate cancer cells in vivo.

Global gene expression analysis on the isogenic Myc-CaP lines revealed several genes differentially expressed in each pairwise comparison. Compared with Vector control, Twist1 overexpression altered the expression of 424 genes. Twist1-F191G overexpression compared against Vector control altered the expression of much fewer genes, 53 genes, and the majority (28/53) of these genes were also observed with Twist1 overexpression. Between Twist1 and Twist1-F191G, 158 genes were altered, of which the majority were also altered in Twist1 (81/158; Fig. 6A and Supplementary Tables S1 and S2). This expression pattern is consistent with Twist1-F191G having a greatly attenuated transcriptional program compared with wild-type Twist1 (Fig. 6B). These global gene expression profiling data are consistent with our promoter reporter assays conducted above suggesting that the Twist box domain is required for the full transcriptional activity of Twist1 (Fig. 2B and Supplementary Fig. S1). These findings are highly suggestive of a transcriptional mechanism for the phenotypic differences observed between prostate cancer cells overexpressing Twist1 and those overexpressing the Twist box mutant.

GSEA (38) was used to identify gene sets which were overrepresented in Twist1 but not Twist1-F191G. Many of the overrepresented gene sets were related to phenotypes which we directly assayed, aggressive cellular behavior and metastasis, and were observed with overexpression of Twist1 but not Twist1-F191G (Fig. 6C). One gene set of interest was directed by the homeobox transcription factor, Hoxa9, which is strongly implicated in leukemogenesis. Furthermore, the Hox homolog HOXA5 had been shown previously to interact physically with Twist and antagonize repression of p53 to genotoxic stressors. Thus, we confirmed our microarray data by qPCR and Western blotting showing that Twist1 overexpression resulted in Hoxa9/HOXA9 overexpression in Myc-CaP and PC3 (Fig. 6D and E; both P < 0.01 by qPCR and Supplementary Fig. S11A and S11B by Western blotting). Twist1 also bound to the Hoxa9 promoter in a region containing canonical E-box sequences as shown by ChIP-qPCR (Fig. 6F). Consistent with our global gene expression data, the Twist box mutant was unable to upregulate the expression of Hoxa9/HOXA9 overexpression in Myc-CaP and PC3 and was similar to Vector control (Fig. 6D and E; both P < 0.05 by qPCR and Supplementary Fig. S11A and S11B by Western blotting). However, the Twist1-F191G mutant was still capable of binding to the Hoxa9 promoter by ChIP-qPCR, suggesting that the Twist box was required for the full transcriptional activity of Twist1 (Fig. 6F and summarized differences between in vitro and in vivo phenotypes of Twist1 and Twist1-F191G in Fig. 6G).

Interestingly, we found that many of the in vitro prometastatic phenotypes of Twist1 overexpression in Myc-CaP cells were significantly blunted following short hairpin RNA (shRNA)-mediated knockdown of Hoxa9 (Fig. 7). Three separate shRNA constructs against Hoxa9 were each able to knockdown Hoxa9 mRNA and protein expression in Myc-CaP cells overexpressing Twist1 (Fig. 7A; P < 0.05 for qPCR). Hoxa9 knockdown in these Twist1-overexpressing cells resulted in a reduction in Twist1-induced cellular migration, invasion, anoikis resistance, and soft agar clonogenicity (Fig. 7B–G; P < 0.05 all at least). Collectively, these data suggest that Twist1 imparts prometastatic phenotypes on prostate cancer cells, in part, by directly upregulating Hoxa9 expression.

Our study shows that Twist1 overexpression in prostate cancer cells induces an EMT phenotype, augments migration, invasion, and resistance to anoikis and metastasis. We show that the highly conserved Twist box domain is required for many of these properties of Twist1 associated with aggressive tumor cell behavior in vitro and most importantly for metastasis in vivo. We also show that the Twist box domain is required for the full transcriptional activity of Twist1 and facilitates these prometastatic cellular functions by directing specific transcriptional programs. We show that Twist1 directly regulates the transcriptional prometastatic target, Hoxa9, which is at least partially required for Twist1-induced prometastatic phenotypes in prostate cancer cells.

The Twist box is highly conserved among vertebrates and is critical for the role of Twist1 in development as shown by inactivating mutations in this region of the human gene resulting in the Saethre–Chotzen syndrome, characterized primarily by craniosynostosis (5, 39). Consistent with humans, the Charlie Chaplin mouse strain with craniosynostosis and hind-limb abnormalities results from a S192P substitution mutation in the Twist box domain (40). Mechanistically, Twist1 binds to the Runx2 transcription factor via the Twist box and inhibits the Runx2 transcriptional program necessary for osteoblast differentiation. Similarly, Twist1 binds Sox9 via the Twist box and inhibits Sox9-dependent transcriptional programs required for chondrocyte differentiation (41). Twist1 may also directly modulate transcription of target genes, and the Twist box was shown to be both necessary and sufficient for this transactivation activity (34). The Twist box transactivation domain likely adopts an α-helical structure, and the three amino acids, Leu-187, Phe-191, Arg-195, are essential for transactivation function and may occupy the same three-dimensional surface of this α-helix. Twist1 has been shown to directly upregulate the expression of several target genes important for cancer progression like Akt2 expression, which enhances cell migration, invasion, and resistance to chemotherapy (42). In agreement with these findings in breast cancer, our expression profiling of Twist1-overexpressing prostate cancer cells were similar to gene signatures consistent with increased cell migration, invasion, and resistance to apoptosis. However, whether the Twist box mediates a transcriptional program required for Twist1-induced metastasis in prostate cancer cells by actively inhibiting another transcription factor or by directly regulating downstream target genes requires further study.

The role of the Twist box in cancer-related functions has only recently been appreciated. In a recent study, the Twist box was required for Twist1 binding to the NF-κB subunit RELA to activate transcriptional activity, increased DNA-binding affinity to the interleukin 8 (IL-8) promoter and transcriptional activation that was required for breast cancer cell invasion in vitro (43). We did not observe direct Twist box-dependent regulation of IL-8 by Twist1 in our system, but in agreement with this study, we did observe Twist box-dependent gene sets consistent with NF-κB–regulated inflammatory genes. The Twist box has also recently been shown to bind p53 and induce destabilization via MDM2-mediated degradation in sarcoma cells (44). This mechanism is separate from Twist1 indirect regulation of p53 by modulating the ARF/MDM2/p53 pathway (45). We do not believe that Twist box-dependent p53 regulation explains the Twist1-induced metastatic phenotypes we observed in prostate cancer cells as PC3 cells are TP53-null and a similar Twist1-F191L substitution mutation in the Twist box did not affect Twist1-p53 interaction (44). Our study findings require validation in other cancer histologies before our results can be generalized further and confirmation using autochthonous transgenic models of tumorigenesis and spontaneous metastasis models is needed. Despite these limitations, our current study confirms the recent in vitro data showing the importance of the Twist box domain for the protumorigenic activities of Twist1. Importantly, our study shows for the first time the requirement of the Twist box for the prometastatic functions of Twist1 in vitro and in vivo.

TWIST1 expression levels seem to be correlated with prostate cancer aggressiveness and factors associated with lethal metastatic disease (Fig. 1; refs. 7, 33). The downregulation of Twist1 in androgen-independent prostate cancer cells increased their sensitivity to anticancer drugs and suppressed their migration and invasion abilities, suggesting Twist1 inactivation as a potential therapeutic strategy (7). TWIST1 expression during postnatal life is restricted tightly to a subpopulation of mesoderm-derived tissues, and limited studies suggest that Twist1 inhibition systemically may be well tolerated (46). Furthermore, our previous study suggested that suppression of Twist1 to physiologic levels in vivo is sufficient for anticancer effects (11). However, the direct inhibition of Twist1 as a therapeutic maneuver still poses a few potential challenges. First, Twist1 is a pleiotropic transcription factor essential for mammalian development (47). Second, bHLH transcription factors have been difficult to target directly with small molecules (48). A solution to these issues is dissecting what are the critical domains of Twist1 and what are the crucial Twist1 downstream transcriptional targets that are required for Twist1-dependent tumorigenicity and prometastatic functions.

Using comparative gene expression profiling of Twist1 and Twist box mutant cells, we discovered Hoxa9 as a novel direct gene target of Twist1 that was required, in part, for many Twist1-induced prostate cancer prometastatic phenotypes in vitro. Although there is a rich literature on the oncogenic role of Hoxa9 in leukemia (49), only one recent report has suggested a role for Hoxa9 in prostate cancer (50). Thus, we have uncovered a novel mechanism involving Twist1 and Hoxa9 oncoproteins collaborating to facilitate prostate cancer progression and metastatic cellular behavior.

In conclusion, these data herein have increased our insight into the structure–function relationships of the Twist1 oncoprotein in cancer and point to the Twist box as a critical domain required for directing transcriptional prometastatic programs in prostate cancer cells. Our findings suggest therapeutic measures against TWIST1-overexpressing prostate cancer cells should be minimally directed against the Twist box domain and Twist1-regulated transcriptional targets such as Hoxa9.

No potential conflicts of interest were disclosed.

Conception and design: S.T. Chettiar, T.F. Burns, R.K. Hales, S.S. An, P.T. Tran

Development of methodology: R.P. Gajula, S.T. Chettiar, S.S. An, P.T. Tran

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.P. Gajula, S.T. Chettiar, R.D. Williams, S. Thiyagarajan, R. Wang, N. Gandhi, A.T. Wild, F. Vesuna, J. Ma, T. Salih, T.F. Burns, C.H. Chung, S.S. An, P.T. Tran

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.P. Gajula, S.T. Chettiar, R.D. Williams, S. Thiyagarajan, K. Aziz, R. Wang, N. Gandhi, A.T. Wild, J. Ma, E. Fertig, S. Biswal, C.H. Chung, R.K. Hales, S.S. An, P.T. Tran

Writing, review, and/or revision of the manuscript: R.P. Gajula, S.T. Chettiar, Y. Kato, K. Aziz, N. Gandhi, A.T. Wild, F. Vesuna, J. Cades, E. Fertig, T.F. Burns, C.H. Chung, C.M. Rudin, J.M. Herman, R.K. Hales, S.S. An, P.T. Tran

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.P. Gajula, S.T. Chettiar, Y. Kato, K. Aziz, N. Gandhi, V. Raman, P.T. Tran

Study supervision: P.T. Tran

This work was funded by RSNA Research Medical Student Grant (A.T. Wild), Johns Hopkins Laboratory Radiation Oncology Training Fellow NIH-T32CA121937 (to R.D. Williams), NIH P50CA103175 and U54CA141868 (to S.S. An), and Phyllis and Brian L. Harvey Scholar Award from Patrick C. Walsh Prostate Cancer Research Fund, DoD Prostate Cancer Physician Research Training Award W81XWH-11-1-0272, ACS Scholar 122688-RSG-12-196-01-TBG, and the NIH P50CA103175 and R01CA166348 (to P.T. Tran).

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