TRIM29 promotes bladder cancer invasion by regulating the intermediate filament network and focal adhesion

Bladder cancer is a common malignancy whose lethality is determined by invasive potential. We have previously shown that TRIM29, also known as ATDC, is transcriptionally regulated by TP63 in basal bladder cancers where it promotes invasive progression and metastasis, but the molecular events which promote invasion and metastasis downstream of TRIM29 remained poorly understood. Here we identify stimulation of bladder cancer migration as the specific role of TRIM29 during invasion. We show that TRIM29 physically interacts with K14 + intermediate filaments which in turn regulates focal adhesion stability. Further, we find that both K14 and the focal adhesion protein, ZYX are required for bladder cancer migration and invasion. Taken together, these results establish a role for TRIM29 in the regulation of cytoskeleton and focal adhesions during invasion and identify a pathway with therapeutic potential.


Introduction
Bladder cancer is the sixth most common cancer in the United States and will cause > 17,000 deaths in 2023 [1].Clinically, bladder cancers are categorized as either non-muscle-invasive (NMI) or muscleinvasive (MI) based on their ability to invade stroma and muscular barriers.NMI bladder cancers are mostly low-grade, multifocal, and frequently recur after surgical removal.Approximately 15% of these NMI bladder tumors will progress to MI bladder cancers.MI bladder cancers in which tumors invade through the basement membrane and migrate into the muscularis propria require more aggressive multimodality therapy with chemotherapy, radiation, and surgery.Despite this aggressive and toxic therapy, up to 50% of patients will have lethal metastatic relapses.Thus, invasive progression determines patient outcomes in bladder cancer.
Despite the clinical importance, the molecular drivers of invasive progression in bladder cancer are poorly understood.We have previously identi ed Tripartite Motif-containing protein 29 (TRIM29), also known as Ataxia Telangectasia Group D Complementing (ATDC), as an important driver of bladder cancer initiation and invasive progression [2,3].TRIM29 is part of the basal gene program which drives tumor progression and metastasis [3].However, the molecular mechanism by which TRIM29 impacts bladder cancer invasion was not previously well understood.
Cell motility plays a key role in cancer invasion and is regulated by a network consisting of highly coordinated cytoskeleton proteins which include actin micro laments and intermediate lament (IF) proteins such as vimentin and keratins.Keratin 14 (K14), one of the components of IF and a marker of basal epithelium, is upregulated in the leading cells of invading breast tumor and plays an important role in regulating invasion [4].We previously reported that TRIM29 and KRT14 are both upregulated by TP63 in invasive basal bladder cancers [3], but how TRIM29 and K14 interact with the cytoskeleton to coordinate invasion remained unclear.
The focal adhesion complex (FA), which serves as an anchor point for cells to attach to the extracellular matrix (ECM), is one of the main structures connecting the ECM to intracellular cytoskeleton networks including actin micro laments, microtubules, and intermediate laments [5,6].FA are dynamic protein complexes required for proliferation, migration, and invasion of cancer cells [7,8].The FA complex develops on the cytoplasmic side of the cell membrane, where integrin receptors cluster.FA complexes are comprised of scaffold or adapter proteins like paxillin (PXN), which recruit downstream signaling factors, including focal adhesion kinase (FAK) [9].Phosphorylation/activation of FAK promotes binding of regulatory proteins such as c-Src, vinculin and talin which leads to polymerized actin assembly and a physical connection between focal adhesion sites and cytoskeleton network, and eventually enhances cell motility and invasion by regulating dynamic rearrangement of the actin cytoskeleton [10,11].
In this study, we use 2D and 3D models of migration and invasion to identify the role of TRIM29 in bladder cancer invasion.We nd that TRIM29 speci cally regulates cell migration by binding to K14 + intermediate laments (IF), regulating formation of K14 + IFs in the invadopodia of bladder cancer cells.
Further, this function regulates the stability of the FA complex during cancer cell migration and invasion.
These results provide a better understanding of the regulation of invasive progression in bladder cancer and identify new potential therapeutic targets to prevent progression to the lethal invasive form of bladder cancer.

Modi ed 2D cell migration (Scratch) assay and phasecontrast microscopy
A coverglass-bottomed 4-well chamber (#155382 Nunc Lab-Tek II, Thermo Fisher Scienti c) was coated with poly-L-lysine (#4832, Sigma-Aldrich, Burlington, MA) and collagen solution (3mg/ml, STEMCELL Technologies, Vancouver, Canada); a cell culture insert (#80209, Ibidi, Gräfel ng, Germany) was placed securely on the coverglass.Cells reaching 80% con uence were trypsinized and transferred into the cell culture insert (3.5 x 10 4 cells/insert chamber).Cells were cultured inside the insert overnight (or until the cells reach 80% or 90% con uence).The insert was removed to start the migration assay.2D migration was observed and recorded by a LSM800 microscope (Zeiss, Oberkochen, Germany) equipped with timelapse imaging ability and a climate control chamber.Images were taken at 10-min intervals and then analyzed by ZEN2 software (Zeiss).Cells on the leading edge were selected and tracked for calculating the velocity of migration.

Immunoprecipitation
Cells cultured on plates reaching 90% con uence were lysed in buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 5% glycerol) for immunoprecipitation (IP).For conventional IP, the lysis buffer contained 0.1% NP40.Cell lysates were centrifuged at 3000 g for 10 min, and then the supernatants were collected.Magnetic beads (10015D, Thermo Fisher Scienti c) were coated with TRIM29 antibody (B-2, Santa Cruz Biotechnology, Dallas, TX) for 6 h at 4°C before adding to cell lysates by following manufacturer's instructions.Antibody-coated bead and cell lysates were mixed in 4°C for overnight.
Beads were washed with cold PBS twice before adding protein sample loading buffer (#1610747, Bio-Rad, Hercules, CA) and heated at 95°C for 5 min.Immunoprecipitated samples were used to perform Western blot or mass spectrometry.

Liquid Chromatography and Mass Spectrometry
The beads were resuspended in 50 µl of 0.1M ammonium bicarbonate buffer (pH ~ 8).Cysteines were reduced by adding 50 µl of 10 mM DTT and incubating at 45°C for 30 min.Samples were cooled to room temperature and alkylation of cysteines was achieved by incubating with 65 mM 2-Chloroacetamide under darkness for 30 min at room temperature.An overnight digestion with 1 µg sequencing grade, modi ed trypsin was carried out at 37°C with constant shaking in a thermomixer.Digestion was stopped by acidi cation and peptides were desalted using SepPak C18 cartridges using manufacturer's protocol (Waters, Milford, MA).Samples were completely dried by using vacufuge.Resulting peptides were dissolved in 9 µl of 0.1% formic acid/2% acetonitrile solution and 2 µl of the peptide solution were resolved on a nano-capillary reverse phase column (Acclaim PepMap C18, 2 micron, 50 cm, Thermo Fisher Scienti c) using a 0.1% formic acid/2% acetonitrile (Buffer A) and 0.1% formic acid/95% acetonitrile (Buffer B) gradient at 300 nl/min over a period of 180 min (2-25% buffer B in 110 min, 25-40% in 20 min, 40-90% in 5 min followed by holding at 90% buffer B for 10 min and equilibration with Buffer A for 30 min).Eluent was directly introduced into Q exactive HF mass spectrometer (Thermo Fisher Scienti c) using an EasySpray source.MS1 scans were acquired at 60K resolution (AGC target = 3x106; max IT = 50 ms).Data dependent collision induced dissociation MS/MS spectra were acquired using Top speed method (3 seconds) following each MS1 scan (NCE ~ 28%; 15K resolution; AGC target 1x105; max IT 45 ms).Proteins were identi ed by searching the MS/MS Human Protein Database (20286 entries; reviewed; downloaded on 06/17/2020) and ltered for high con dence proteins using Proteome Discoverer (v2.4,Thermo Scienti c).Search parameters included MS1 mass tolerance of 10 ppm and fragment tolerance of 0.2 Da; two missed cleavages were allowed; carbamidimethylation of cysteine was considered xed modi cation and oxidation of methionine, deamidation of aspargine and glutamine were considered as potential modi cations.False discovery rate (FDR) was determined using Percolator and proteins/peptides with a FDR of = 1% were retained for further analysis.

Immuno uorescence staining and imaging
Cells were seeded on poly-L-lysine-and collagen-coated coverglass.After designated treatment and time of incubation, cell samples were xed by 4% paraformaldehyde in PBS for 10 min, then permeabilized with 0.5% Triton X-100 in PBS for 5 min.After thoroughly washing with PBS, samples were submerged in blocking solution (PBS containing 5% BSA) for 1 h at room temperature.After blocking, the samples were covered with primary antibody solution (designated primary antibody in PBS, 1:50 dilution) at room temperature for 16 h, followed by washing thoroughly with PBS.Then the samples were incubated in secondary antibody solution (1:300 dilution) for 1 h at room temperature and washed thoroughly with PBS before staining for nuclei with Hoechst 33342 solution for 5 min at room temperature.Samples were washed thoroughly again with PBS before mounted on glass slides with 30 µl mounting media.Images were obtained with confocal microscope Zeiss LSM800.

Realtime Imaging of K14 + intermediate laments during cell migration
UM-UC5 or UM-UC14 cells were transfected with mCherry-labeled K14 (#55066, Addgene).Cells were cultured on collagen type I-coated coverglass chamber slide within a culture insert.After removal of the insert, LSM800 confocal microscope (Zeiss) was used to visualize the area covered by the migrating cells.Time-lapse imaging was conducted at 3-minute intervals using a 63X oil objective.

Quanti cation of the number and size of focal adhesion plaques
Con uent bladder cancer cells were trypsinized and cultured in ultra-low attachment 6-well culture plate (0.5 x 10 6 cells/well) for 24 h to form cancer spheroids.Spheroids were transferred to a 12-well plate with one poly-L-lysine-and collage type I-coated (3mg/ml, STEMCELL Technologies) coverglass placed in each well.Spheroids were allowed to attach and expand on coverglass for 48h at 37°C with 5% CO 2 supply.Samples were xed and stained with anti-ZYX or anti-PXN antibodies according to immuno uorescence staining procedures described above.Images were taken by a LSM800 confocal microscope (Zeiss).The cells located on the leading edge of the expanded spheroids were selected manually by using ZEN2 software.The size and number of the ZYX or PXN + focal adhesion sites, were quantitated using ZEN2.Only focal adhesion sites ≥ 0.02 µm 2 were examined.Site number/cell was recorded.

Realtime Analysis of focal adhesion dynamics
TRIM29 wildtype or KO bladder cancer cells (UM-UC5 and UM-UC14) were transfected with a plasmid expressing mCherry-tagged PXNn (#50526, Addgene) before culturing on collagen type I-coated coverglass chamber with culture inserts.The insert was removed to allow cells to migrate for 72 h, and the area of the migration was imaged with a LSM800 confocal microscope (Zeiss).Time-lapse imaging was carried out with 3-min intervals and seven slices of z stack (spanning height of 70 nm) by using 63X oil objective.The quanti cation of focal adhesion dynamics, including the rate of FA assembly (Ka) and disassembly (Kd), was performed according to the methods described in [12].

Data Availability
All data presented in the Results were generated by the authors and are available on request.

Results
Loss of TRIM29 blocks bladder cancer migration.The tripartite motif gene, TRIM29, is required for invasion in bladder cancer [2,3] and many other malignancies [13,14].Invasion is a complex process that involves degradation of extracellular matrix barriers, cellular detachment and reattachment, and increased motility [15,16].To determine which of these aspects of invasion were regulated by TRIM29 in bladder cancer, we examined invasion and migration using transwell, 3D collagen tumor spheroid invasion and modi ed scratch assays.TRIM29 was knocked down (TRIM29-KD) with shRNA in UM-UC13 or knocked out with CRISPR in UM-UC14 and UM-UC5 as previously described (Fig. 1A) [3].As expected, TRIM29 knockdown (TRIM29-KD) in UM-UC13 and TRIM29 knockout (TKO) in UM-UC5 and UM-UC14 signi cantly reduced transwell invasion (Fig. 1B) and 3D tumor spheroid invasion (Supplemental Fig. 1).To determine whether TRIM29 was speci cally required for cancer cell migration, we performed a modi ed scratch assay in which cells were cultured on collagen type I-coated cover glass, a scratch is created by insert removal, and individual cell migration velocity was quanti ed by time-lapse microscopy.TRIM29-KO (UM-UC5, UM-UC14) or TRIM29-KD (UM-UC13) signi cantly reduced migration of individual cancer cells (Fig. 1C and 1D).Re-expression of TRIM29-FLAG in the TRIM29-KO cells (UM-UC5 and UM-UC14) rescued the cell migration ability disrupted by knockout of TRIM29 (Fig. 1D, Supplemental Fig. 2).These results establish that TRIM29 is required for bladder cancer cell migration during invasion.
TRIM29 is part of a protein complex including intermediate lament, motor and focal adhesion proteins.
TRIM29 has previously been demonstrated to exert cellular effects by binding and sequestering proteins and regulating ubiquitination and protein stability [2,[17][18][19][20].To comprehensively identify the interactome of TRIM29 in bladder cancer and identify the mechanism by which TRIM29 promotes bladder cancer migration and invasion, we performed immunoprecipitation (IP) of TRIM29 and liquid chromatography and mass spectrometry (LC/MS) in UM-UC5 and 9 bladder cancer cell lines to identify proteins present in the immunocomplex with TRIM29.UM-UC5 and UM-UC9 TRIM29-KO cells which lack TRIM29 expression were subjected to TRIM29 IP and LC/MS to control for nonspeci c pulldown and act as a negative control.We identi ed 1125 proteins in UM-UC5 and 270 proteins in UM-UC9 which were selectively immunoprecipitated in TRIM29 WT but not KO samples (Supplemental Table 1).144 of these proteins were detected in both UM-UC5 and UM-UC9.As expected, TRIM29 was one of the most enriched proteins in both cell lines.To identify the functional classes of the 1125 and 270 proteins selectively co-IP'd with TRIM29 in UM-UC5 and UC9, we subjected each gene list to KEGG pathway enrichment analysis.
For both UM-UC5 and UC9, there was striking enrichment in genes related to Regulation of Actin Cytoskeleton and Focal Adhesion in our TRIM29 co-IP protein complexes (Supplemental Table 2).Within these pathways, the proteins involved in regulation of focal adhesion and regulation of the actin cytoskeleton (MYH9, MYO1C) have previously been shown to regulate cell adhesion, migration and invasion [21].Additionally, we found enrichment in numerous intermediate lament proteins (K5, K6A, K8, K9, K10, K18).These results suggested that TRIM29 forms protein complexes with focal adhesion, actin cytoskeleton, and intermediate lament proteins, suggesting a potential means whereby TRIM29 regulates cancer migration and invasion.
TRIM29 Regulates K14 + IF in Invasive and Migratory Cells.KRT14 is transcriptionally regulated by TP63 [22] and is upregulated in invasive leader cells in breast cancer by TP63 [4].We have previously shown that TP63 regulates transcription of TRIM29 and KRT14 in basal bladder cancer, that TRIM29 is upstream of K14 and that both are required for bladder cancer invasion [3].Since TRIM29 forms a protein complex with IF proteins in bladder cancer cells in our LC/MS screen and regulates keratin distribution in squamous cell carcinoma [23], we hypothesized that TRIM29 might speci cally localize to K14containing IF to regulate migration and invasion.To examine this, we generated bladder cancer spheroids in suspension conditions as previously described and embedded them in type I collagen on coverglass [3].Spheroids were cultured for 48 h, xed and subjected to immuno uorescent staining for TRIM29, actin and K14.Both TRIM29 and K14 were selectively upregulated in the cells in the invasive component of the bladder cancer spheroids (Fig. 2A, region outside of dotted line).These results suggest that TRIM29 is selectively upregulated with K14 in the invading tumor cells.
TRIM29 localizes to lamentous structures and has previously been reported to interact with the IF, Vimentin [24].We observed that multiple keratins were part of the TRIM29 immunocomplex in bladder cancer cells (Supplemental Table 1), thus we hypothesized that TRIM29 might localize to IFs during invasion.To determine this, we performed immuno uorescent staining for TRIM29, K14, and actin in multiple bladder cancer cells.We chose K14 and not other keratins identi ed in LC/MS screen because of our prior data linking TRIM29 function to K14 [3].TRIM29 and K14 strongly co-localized to the IF structures observed in the invasive cells of multiple bladder cancer cell lines (colocalization between K14 and TRIM29 shown in white, Fig. 2B).Interestingly, although TRIM29 and K14 were always present in contiguous IF structures throughout the cytoplasm, the peripheral, membrane proximal regions in lamellipodia showed the highest overlap (arrows, Fig. 2B).Given that TRIM29 and K14 were selectively upregulated in migratory/invasive cells (Fig. 2A), these results suggested the TRIM29 and K14 might both be present in the same protein complex during migration.
To determine the role of TRIM29 in K14 lament dynamics in migratory bladder cancer cells, we transduced UM-UC5 and UM-UC14 cells with a K14-mCherry fusion protein (Fig. 2C).To determine if K14 and TRIM29 were part of the same protein complex in these cells, we performed co-immunoprecipitation (IP) using an anti-mCherry antibody.We found that IP of mCherry-K14 resulted in co-IP of TRIM29 in UM-UC5 and UM-UC14 cell lines but not in the control cells lacking K14-mCherry (Fig. 2C).Likewise, IP of TRIM29 also resulted in co-IP of K14-mCherry (Supplemental Fig. 3) establishing that TRIM29 and K14 are part of the same protein complex.
Next, to assess the role of TRIM29 in regulation of K14 dynamics during migration, we observed migrating WT and TRIM29-KO UM-UC5 and UM-UC14 cells expressing K14-mCherry (Fig. 2D-F Supplemental Video 1-4).In TRIM29 WT cells, the migratory cells formed a rigid, symmetrical, and compact cell structure with lamentous K14 distributed evenly through the cytoplasm (Fig. 2D, Supplemental Video 1 and 3).In contrast, TRIM29-KO cells displayed irregular elongated morphologies and K14 did not form regular lamentous structures (Fig. 2D, Supplemental Video 2 and 4).Further, examination of migration in TRIM29 WT and KO cells using time lapse imaging demonstrated that while TRIM29 WT cells showed well organized K14 + laments and e cient cell migration, TRIM29-KO cells lacked organized K14 laments and demonstrated disordered migration (Fig. 2E-F, Supplemental Video 1-4).These results suggest that loss of TRIM29 destabilizes K14 + intermediate laments, changes the morphology of invasive cells and impairs migratory ability.
Mechanism of TRIM29 regulation of K14.We next sought to determine the mechanism by which TRIM29 regulates K14 + IF during invasion.FAM83H has been proposed to bind to TRIM29 and K14 and to regulate K14 distribution in other types of cancer [23].We therefore hypothesized that TRIM29 regulation of K14 might involve interaction with FAM83H.Interestingly, while FAM83H was expressed in UM-UC5 and UM-UC14 cancer cells, it did not co-IP with K14 or TRIM29 suggesting that it was not part of the TRIM29-K14 complex in bladder cancer (Fig. 2C, data not shown).
TRIM29 has also been shown to facilitate ubiquitination and degradation of innate immune proteins during viral infections [19,25].To determine if K14 was a target of TRIM29-mediated ubiquitination, we treated WT and TRIM29-KO UM-UC5 and UM-UC14 cells -with either vehicle or MG132 to block proteasomal degradation of ubiquitinated proteins.While we did observe signi cant differences in total ubiquitination in TRIM29 WT and KO cells, we did not observe either mono or poly-ubiquitinated K14 (Supplemental Fig. 4, data not shown) suggesting that TRIM29-mediated ubiquitination does not contribute to its regulation of K14.K14-dependent disul de bonds mediate some functions of K14 [26].To determine if the TRIM29-K14 protein interaction depended on K14 disul de bonds, we treated lysates with TCEP, a reducing agent which disrupts disul de bonds, and performed IP of K14.We found that the TRIM29 interaction with K14 was reduced by TCEP treatment (Fig. 2C), suggesting that TRIM29 presence in the K14 protein complex was in uenced by disul de bond presence.Taken together, these results indicate that TRIM29 is part of a disul de-dependent protein complex with K14.
TRIM29 and K14 Regulate Focal Adhesions During Invasion.Keratin + IF regulate focal adhesion stability and cellular migration [21] and proteins associated with focal adhesions were pulled down with TRIM29 in UM-UC9 and UM-UC5 cells.Based on these results, we hypothesized that the TRIM29-K14 interaction regulated migration via effects on focal adhesion formation or turnover.To examine this, we performed staining for TRIM29, PXN, zyxin (ZYX-a member of the focal adhesion complex) and actin in our invasive bladder cancer spheroids and found that TRIM29 + IF terminated in PXN + and ZYX + FA sites in the migratory bladder cancer cells (Fig. 3A -D).These focal adhesions sites were concentrated in lopodia and lamellipodia (arrows) on the leading edge of migratory cancer cells.To determine if TRIM29 was required for the formation of these FAs during invasion, we examined staining in UM-UC5 and UM-UC14 TRIM29-KO cells and found that TRIM29-KO signi cantly reduced ZYX + and PXN + focal adhesions (Fig. 4A -E, Supplemental Fig. 5).These results suggest that TRIM29 is involved in FA regulation during bladder cancer cell migration.TRIM29 Regulates Focal Adhesion Stability During Cancer Migration.FA presence during cancer cell migration is dependent on the rate of FA formation and dissolution.Since K6 + IF are known to regulate FA formation and resolution [21] and since TRIM29 was found to be part of the focal adhesion protein complex, we hypothesized that TRIM29 might regulate focal adhesion stability during invasion and migration.To examine this, we measured focal adhesion dynamics in WT and TRIM29-KO bladder cancer cells expressing a mCherry-tagged PXN construct in a migration assay.As expected, mCherry + FA were visible at the leading edge of invasive cells during time-lapse microscopy (Fig. 5A).In WT cells, the focal adhesion sites rst appeared at the leading edge of cells and became larger as they moved gradually toward the posterior edge of the cells, but the foci in TRIM29-KO were present for a shorter amount of time and dispersed more quickly (Supplemental video 5-8).To quantify this process, focal adhesion sites from the time lapse images were randomly selected, mCherry signal was quanti ed and analysis was performed as previously published [21].By analyzing the rates of assembly (Ka) and disassembly (Kd) of each focal adhesion site, we found that the rate of disassembly of focal adhesion is higher in TRIM29-KO cells, whereas the assembly rate was not different between TRIM29-KO and WT (Fig. 5B).These results suggest that TRIM29 regulates FA stability during invasion and migration.K14 is Required for TRIM29 Regulation of FA Stability and Promotion of Bladder Cancer Migration and Invasion.Based on the results that TRIM29 was required to stabilize K14 + IF and focal adhesions during bladder cancer migration and invasion, we hypothesized that TRIM29-mediated stabilization of FA required K14.To test this hypothesis, we knocked-down KRT14 in our TRIM29-KO UM-UC14 cells with or without TRIM29 re-expression (Supplemental Fig. 2) and measured ZYX + and PXN + FA sites in migrating cells.TRIM29 re-expression allowed robust recovery of ZYX + and PXN + focal adhesion sites, but this effect was abrogated by KRT14 knock-down (Fig. 6A & B).These results suggested that TRIM29's promigratory phenotype would require both KRT14 and ZYX.To test this, we performed RNAi-based gene knock-down of KRT14 or ZYX in UM-UC10 and UM-UC14 bladder cancer cell lines with or without TRIM29 expression.Cell migration was then measured by modi ed scratch assay and invasion was measured using a transwell assay.TRIM29 expression promoted increased cancer cell migration and invasion (Fig. 6C & D) [4].Further, knockdown of KRT14 or ZYX expression blocked TRIM29-induced cell migration and transwell invasion (Fig. 6C & D).Taken together, these results demonstrate K14 is required for TRIM29-mediated stabilization of FA and that both KRT14 and ZYX, are required for TRIM29-mediated migration and invasion.

Discussion
Invasive progression is the most important biological factor which determines bladder cancer clinical outcomes.We have previously shown that bladder cancer invasion is driven by TP63-mediated upregulation of TRIM29 and KRT14 [3].However, the mechanism whereby TRIM29 and K14 promote invasive progression and metastasis was unclear.In this study, we demonstrate that a primary function of TRIM29 during invasion is the regulation and promotion of tumor cell migration.Mechanistically, TRIM29 speci cally regulates tumor migration by physical association with the K14 + intermediate laments in invasive bladder cancer cells.Further, the physical association of TRIM29 with K14 and focal adhesion sites appears to stabilize these structures and alter cellular morphology, promoting a proinvasive, pro-migratory phenotype (Fig. 7).These results establish a new role for TRIM29 in the regulation of cytoskeletal functions and focal adhesion interaction with the extracellular matrix, areas where TRIM29 has not been previously implicated.
The basal molecular subtype of bladder cancer is associated with worse survival and poorer outcomes for patients [27,28].Activation of a TP63-mediated basal gene program in tumor cells has been linked to tumor invasion and metastasis in breast and bladder cancers by our group as well as others [3,4].Here we demonstrate that this basal gene program which includes upregulation of TRIM29 and KRT14, can occur selectively in invasive bladder cancer cells and speci cally promotes cancer cell motility by altering the physical structure of the invasive cell and its attachments to ECM.These results establish novel mechanisms that contribute to the lethal phenotypes seen in basal subtype bladder cancer.They also suggest that treatments which disrupt focal adhesion functions may be an attractive approach for bladder cancer patients with basal subtype tumors which have poor outcomes and for which treatment options are limited.
The role of intermediate laments and focal adhesion complex proteins in normal cell motility and in cancer cell invasion and metastasis is complex [5,[29][30][31][32][33]. Intermediate laments are known to interact directly with and regulate the focal adhesion complex [5].Likewise, the focal adhesion complex, which includes integrins, adaptor proteins such as PXN and ZYX, and kinases like FAK and c-Src, is a known subject to serine and tyrosine phosphorylation which regulate focal adhesion complex formation and dissolution during cell migration [9,34].Since we nd that TRIM29 modulates focal adhesion dissolution, we hypothesize that it may do so by promoting PXN phosphorylation which has been shown to govern focal adhesion stability.Alternatively, TRIM29 has also been shown to harbor ubiquitin ligase function [19,20] and it is conceivable that TRIM29 directly ubiquitinates K14 or one or multiple components of the focal adhesion complex leading the phenotypes observed in this study.Despite this notion, we were unable to detect ubiquitination of either K14, PXN or ZYX in multiple experiments suggesting that TRIM29 does not directly ubiquitinate K14, PXN or ZYX (Supplemental Fig. 4 and data not shown).
High levels of FAK expression often correlate with invasiveness in cancer cells [35], and tumors with high c-Src activity also correlate with malignant potential [36].In bladder cancer, FAK and Src activity have been linked to survival, invasive progression and migration [37][38][39] and PTEN, a downstream target of TRIM29 [2], also has been shown to impact FAK and AKT phosphorylation [40].Our results suggest that a key function of TRIM29 is to stabilize the focal adhesion complex and it is therefore likely that this activity also impacts FAK and c-Src signaling mediating its effect on tumor migration and invasion.Further, if true, this suggests that kinase inhibition of FAK or c-Src may be an attractive therapeutic strategy in patients with invasive basal subtype bladder cancers.

Figure 5 The
Figure 5