A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Bladder Cancer Patients

Author Manuscript Published OnlineFirst on February 24, 2020; DOI: 10.1158/1078-0432.CCR-19-2912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Bladder Cancer Patients.

Vinata B. Lokeshwar1,♠,, Daley S. Morera1,♠, Sarrah L. Hasanali1, Travis J. Yates2,+, Marie C. Hupe3, Judith S. Knapp3, Soum D. Lokeshwar4, Jiaojiao Wang1, Martin Hennig3, Rohitha Baskar1, Diogo O. Escudero5,+, Ronny R. Racine6,+, Neetika Dhir6,+, Andre R. Jordan1,2, Kelly Hoye2,+, Ijeoma Azih7, Murugesan Manoharan8, Zachary Klaassen9, Sravankumar Kavuri10, Luis E. Lopez1, Santu Ghosh11, Bal L. Lokeshwar12

Departments of Biochemistry and Molecular Biology (1); Clinical Trials Office (7), Surgery, Division of Urology (9); Pathology (10); Department of Population Health Sciences (11), Georgia Cancer Center (12), Medical College of Georgia, Augusta University, Augusta, GA, 1410 Laney Walker Blvd., 30912, USA Sheila and David Fuente Graduate Program in Cancer Biology (2), Honors Program in Medical Education (4), Molecular Cell and Developmental Biology Graduate Program (5), Department of Urology (6) University of Miami-Miller School of Medicine, Miami, 1600 NW 10th Avenue, Florida, 33136, USA

Department of Urology, University-Hospital Schleswig-Holstein, Campus Luebeck, Luebeck, Germany (3)

Division of Urologic Oncology Surgery, Miami Cancer Institute, Baptist Health South Florida, Miami, Florida (8)

♠: Contributed equally and are joint first authors

Running title: HYAL4-V1 Chase in Bladder Cancer

*Address for Correspondence:

Vinata B. Lokeshwar, Ph.D. Professor and Chair; Department of Biochemistry and Molecular Biology Medical College of Georgia, Augusta University 1410 Laney Walker Blvd., Room CN 1177A, Augusta, GA 30912-2100 Office: (706) 721-7652; Fax: (706) 721-6608; E-mail: [email protected]

+Present address: Travis Yates: QualTek Molecular Laboratories, King of Prussia, PA; Diogo Escudero: George Washington University Kelly Hoye: University of North Carolina Lineberger Comprehensive Cancer Center; Ronny Racine: Astellas Institute for Regenerative Medicine (AIRM); Neetika Dhir: deceased.

The authors declare no competing interests.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 24, 2020; DOI: 10.1158/1078-0432.CCR-19-2912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abbreviations used: aa: amino acid(s); ALDH: aldehyde dehydrogenase 1; BC: bladder cancer; Chase: chondroitinase; CM: conditioned medium; CS: chondroitin sulfate; CSC: cancer stem cell CSPG: chondroitin sulfate proteoglycan; EV: empty vector; HG: high-grade; LG: low-grade; MIBC: muscle invasive bladder cancer; NBL: normal bladder; sCD44: soluble CD44; stub: Neoepitope generated after chondroitin-6-sulfate chains are removed from a CSPG; V1: splice variant of HYAL- 4; V1-FL: V1-floating; Wt: wild type

ABSTRACT Purpose: Poor prognosis of patients with muscle-invasive bladder cancer (BC) that often metastasizes drives the need for discovery of molecular determinants of BC progression.

Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in BC.

Experimental design: In clinical specimens HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, immunohistochemistry and/or immunoblotting; a novel assay measured

Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three BC cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder.

Results: HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In BC tissues, V1 levels associated with metastasis and cancer-specific- survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from

CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing BC cells formed tumors at low-density and formed metastatic bladder tumors when implanted orthotopically.

Conclusion: Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant BC and potential predictor of outcome in BC patients.

TRANSLATIONAL RELEVANCE

Notwithstanding cystectomy, 50% or more of patients with muscle invasive bladder cancer

(MIBC) develop metastasis. With a dismal 14-month median survival despite chemotherapy, early stratification of BC patients who have a higher risk of developing metastasis is pivotal for improving clinical outcome. Currently, no predictive biomarkers are incorporated into the clinical management of BC patients. The discovery of HYAL4-V1, the first human chondroitinase

(Chase), establishes V1-Chase as a potential independent predictor of metastasis and cancer- specific mortality following cystectomy. The high-throughput Chase and q-PCR assays demonstrate the potential for clinical translation of V1-Chase as a prognostic biomarker.

Identification of V1-Chase as a molecular driver of malignant transformation of bladder uroepithelium and metastasis reveals the “why” and “how” V1-Chase is a potential prognostic biomarker. The results demonstrated here also reveal that V1-Chase, a previously untapped class of molecules, could pave the way for developing strategies to target V1-Chase for the treatment of metastatic BC.

INTRODUCTION

Bladder cancer is a common cancer of the urinary tract with two distinct characteristics, frequent recurrence and heterogeneity in tumor progression. Ninety percent of bladder tumors arise from malignant transformation of the urothelial lining. For bladder tumors, grade indicates invasive potential, while stage indicates depth of invasion. Low-grade tumors are confined to the mucosa (stage Ta) and rarely invade the lamina propria (stage T1). However, high-grade tumors, if not detected early, will present as muscle invasive bladder cancer (MIBC). This results in about 2/3rd of high-grade BC patients presenting with MIBC at initial diagnosis. MIBC is associated with poor prognosis; 50% of these patients develop metastasis within two years.

Although the standard systemic cisplatin-based chemotherapy for metastatic BC yields a reasonable initial response, the majority of these responses are only partial, resulting in a median survival of 14 months. Since the efficacy of the standard systemic chemotherapy has plateaued and the response rate for newer immunotherapy and checkpoint inhibitors is 30% or less, molecular profiling holds the promise of individualizing treatment for MIBC patients (1,2).

Discovery of molecular drivers of BC progression could improve the clinical management of patients with MIBC through better prognostic predictions and potential targeted treatments.

Molecular subtypes of MIBC were one such discovery; however, we recently reported that routine pathology parameters (e.g. grade, lymph node invasion) are more accurate prognostic predictors than the subtypes (3). Therefore, there is still a major unmet clinical need for newly discovered molecular drivers of BC.

Proteoglycans regulate normal physiology and disease processes, including cancer growth and metastasis (4). Chondroitin sulfate proteoglycans (CSPGs), such as Versican,

Biglycan, and Decorin, regulate tumor growth, disease progression, and chemoresistance (5).

CD44, a stem cell marker and hyaluronic acid receptor, is post-translationally modified by

addition of chondroitin sulfate (6-12). Furthermore, our published studies show that CD44- hyaluronic acid signaling in bladder and prostate carcinomas promotes tumor growth, metastasis, and angiogenesis, and can be targeted for therapy (13-15). Although CSPGs and their functions in various cancers are well-studied, a naturally occurring functional eukaryotic chondroitinase (Chase) – an enzyme that removes chondroitin sulfate from CSPGs – has not been identified in any eukaryotic biological system, whether normal or disease condition (benign or malignant).

Our laboratory was one of the first to connect hyaluronidase(s) to cancer biology and to establish it as a driver of BC growth and progression (16-18). The hyaluronidase (HYAL) family of enzymes has six members occurring as two gene clusters on two chromosomes. HYAL-1,

HYAL-2, and HYAL-3 are present on chromosome 3p21.3, whereas HYAL-4, PH20 (testicular hyaluronidase) and HYAL-P1 (a pseudogene) localize to chromosome 7q31.3 (19,20). With the exception of HYAL-4 and HYAL-P1, members of this family degrade hyaluronic acid. Both

HYAL-1 and PH20 are well-studied members of this family. We established HYAL-1 as a molecular determinant of BC and prostate cancer growth, invasion, and angiogenesis, as well as a potential diagnostic marker and prognostic predictor of disease progression and cancer- specific survival (16-18). HYAL-4 is included in the hyaluronidase family based on sequence homology; however, recombinant HYAL-4 has been shown to degrade chondroitin sulfate

(21,22). Based on the cDNA sequence, wild type (Wt) HYAL-4 protein is predicted to have 481 amino acids (aa) and to be anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI) anchor (21,22). Kaneiwa et al expressed a recombinant soluble form of HYAL-4 in

COS-7 cells, in which the N-terminal 33 aa were replaced by a cleavable leader sequence and the last C-terminal 19 aa (GPI-anchor region) were deleted. This recombinant HYAL-4 protein preferentially degraded chondroitin sulfate (21,22). However, except for two GWAS studies,

HYAL-4 expression, Chase activity of the native HYAL-4 protein, and/or HYAL-4/Chase functions have not been reported in any biological system (23,24).

In this study, we demonstrate that while expression of wild-type (Wt) HYAL-4 is undetectable, expression of a HYAL-4 splice variant, HYAL-4 V1 (V1), and its Chase activity are up-regulated in BC. Moreover, V1 expression potentially predicts clinical outcome. Using multiple immortalized urothelial and BC cell systems, overexpression/knockdown approaches, and designing a novel assay for measuring Chase activity, we demonstrate that V1 has Chase activity and induces an invasive, stem cell phenotype. We also demonstrate that CD44 is the primary substrate of V1’s Chase activity. Furthermore, the malignant phenotype induced by V1 is mediated through CD44. V1 expression induces invasive and highly angiogenic tumor formation in an immortalized urothelial cell xenograft and in a BC xenograft, at low cell density.

Moreover, V1 expression induces bladder tumor formation and spontaneous metastasis in an orthotopic model.

Methods

Clinical specimens: Eighty-three bladder specimens (52 BC and 31 normal bladder) were obtained at the University of Miami, Miller School of Medicine. We obtained informed consent from the patients. The study was conducted in accordance with recognized ethical guidelines

(e.g., Declaration of Helsinki, CIOMS, Belmont Report, U.S. Common Rule) and the study was approved by an institutional review board. De-identified specimens and de-linked data were transferred to Augusta University under an approved protocol. Specimen characteristics and associated clinical information are described in Table S1.

We obtained informed consent from the patients. The study was conducted in accordance with recognized ethical guidelines (e.g., Declaration of Helsinki, CIOMS, Belmont Report, U.S.

Common Rule) and the study was approved by an institutional review board.

Cells and reagents: BC cell lines – HT1376 and T24 were purchased from American Type

Culture Collection. 253J cells were provided by Dr. Colin Dinney (MD Anderson Cancer Center).

Immortalized normal bladder epithelial cell line Urotsa was provided by Dr. Donald Sens,

University of North Dakota. Bladder cell lines were authenticated and tested for mycoplasma

(latest testing date: March 2019) by Genetica DNA Laboratories Inc., Cincinnati OH. All experiments were conducted within 6 – 10 passages. Reagents and antibodies are described previously and in Table S2 (15,25). Cells were cultured in RPMI 1640 plus fetal bovine serum and gentamicin. For the collection of serum-free cell conditioned media (CM), 60%-70% confluent cultures in 12-well plates were incubated in RPMI 1640 + ITS supplement (insulin, transferrin, selenium) + gentamicin. Following 48 h incubation, CM were collected. Results of all assays using CM were normalized to cell number.

Tissue survey panel for HYAL-4 expression: TissueScan Cancer Survey Panel 4x96 that includes 381 cDNAs from both normal and tumor was purchased from Origene (Cat #

CSRT303; 5 identical sets for a total of 20 plates). Reverse transcription quantitative PCR (RT- qPCR) was performed using HYAL-4 common primer as described in Table S3.

Cloning and generation of stable transfectants: EV, Wt and V1 transfectants: Total RNA was isolated from high-grade bladder tumor tissues and HT1376 cells and subjected to RT-PCR using HYAL-4 cloning primers (Table S3). By a two-step cloning protocol, Wt and V1 cDNAs were cloned into the PQCXIH retroviral expression vector that added a 3X-FLAG tag at the C- terminus of the Wt and V1 proteins. EV is the empty vector. Bladder cell lines were stably

transfected with EV, Wt or V1 constructs by retroviral infection and the transfectants were selected in hygromycin.

HYAL-4 shRNA transfectants: HYAL-4 shRNA constructs were purchased from Origene.

HT1376 and T24 cells were transfected with HYAL-4 shRNA #1 (sh#1), shRNA # 2 (sh#2) or the control (Ctr) shRNA constructs (please see Table S3 for sequences). siRNA transfection: HT1376 V1 transfectants were transiently transfected with CD44 siRNA as described previously (15,25). Transfectants were analyzed in different phenotypic and biochemical assays, 48 to 72 h post transfection.

Analysis of transcript expression: Total RNA isolated from the transfectants was subjected to

RT-qPCR for the analysis of specific transcripts using gene-specific primers described in Table

S3. The transcript levels were calculated as (1/2ΔCq) x 100; ΔCq = Cq (transcript) – Cq (β-actin).

Immunoblot and immunoprecipitation analyses: Freshly frozen bladder tissues (30 – 50 mg) were homogenized on ice in an ice-cold homogenization buffer (5 mM Hepes pH 7.2, with protease inhibitors). The tissue extracts were clarified by centrifugation at 14,000 rpm for 20 minutes. Tissue homogenates and cell lysates were subjected to immunoblotting for various proteins. All antibodies used for immunoblotting are described in Table S2. Samples were normalized based on equal cell number, total protein, and Actin; protein concentration was measured using DC™ Protein Assay from Bio Rad. Identical protein/cell normalized samples were analyzed for experimental and loading control blots. In each blot, all sample lanes were run on the same gel with the same exposure time; a gap denotes those samples that were not contiguous within the gel. Immunoprecipitation of cell lysates and CM using anti-FLAG mouse monoclonal antibody or mouse IgG was performed using a procedure described before (26).

Immunoprecipitates were immunoblotted using a HYAL-4 antibody either custom-synthesized

(custom Ab) or from Sigma-Aldrich (Sigma Ab). HT1376 V1 CM were also similarly immunoprecipitated with an anti-stub mouse antibody or control antibody (mouse IgG), followed

by immunoblotting with a peroxidase-conjugated anti-CD44 antibody. This procedure involved cell lysis in 1% sodium dodecyl sulfate (SDS) and immunoprecipitation in a buffer with 0.1% final SDS concentration, as described before (26). The initial use of 1% SDS breaks protein complexes and the presence of 0.1% SDS during immunoprecipitation avoids co- immunopreciptation of any complexed proteins.

Chase activity ELISA-like assay: The following biotinylated chondroitin sulfate (CS) derivatives were initially tested in the assay: chondroitin-4-sulfate (CS-A), chondroitin-6-sulfate

(CS-C), chondroitin-2,6-sulfate (CS-D) and CS from shark cartilage. Specifically, biotinylated CS

(1 - 5 µg/well) was linked to 96-well plates (27-30). The plates were incubated with tissue extracts or cell CM in an acetate buffer at 370 C for 16-18 h. Following incubation, biotinylated

CS remaining on each well was determined as described before (31). Chase activity in tissue extracts and cell CM was calculated from a standard graph of O.D. 405 nm versus Chase ABC

(mU/well. Chase activity was normalized to either protein concentration (mg/ml for tissue extract) or cell number (for CM).

Phenotypic readout assays: Proliferation: Transfectants (1.5x104 cells/well) were cultured in growth medium and cell proliferation was assayed by counting viable cells (Trypan blue staining) every 24 h.

Spheroid growth: Transfectants (500 cells) were cultured in MammoCult™ Human medium

(Stem Cell Technologies) on low-attachment plates and the spheroids were photographed after

7 days.

Soft agar assay: Cells (5x103) were seeded in soft agar (2X RPMI 1640 + noble agar (0.6%) on a layer of 2X RPMI 1649 + 1% noble agar). Following incubation for 21 days, Colonies were stained with crystal violet and quantified by light microscopy.

ALDH activity assay: Transfectants were stained with ALDEFLUOR kit (Stem Cell

Technologies) as per the manufacturer’s instructions, followed by flow-cytometry.

Invasion and chemotactic motility: Matrigel™ invasion and chemotactic motility were assayed in two chamber transwells with 8-µm inserts as described previously; for invasion assays, the top well was coated with Matrigel™. Incubation times for motility and invasion assays were 18 h and

48 h, respectively (15,25). In a scratch wound assay, transfectants were cultured in 0.1% FBS containing medium 18 h prior and during wound closure. The wound closure was photographed and calculated as described before (15).

Xenograft studies: Subcutaneous models: All studies on mice were conducted using a protocol approved by the Institutional Animal Care and Use Committee. T24 (1.5x103) or Urotsa

(2x106) transfectants were injected with 30% or 50% Matrigel™ on the dorsal flanks of 7-8 week old male NOD-scid IL2Rgnull mice. HT1376 (1x106) transfectants were injected in athymic mice

(HT1376) with 50% Matrigel. Once palpable, tumor volume was measured twice weekly using calipers.

Orthotopic model: Luciferase expressing HT1376 transfectants (EV, Wt, V1) were intravesically implanted into the bladders of 7-8 week old female athymic mice (4/group); to facilitate tumor cell implantation, bladders were intravescially treated with HCl (0.1 N) prior to tumor cell implantation. Tumor cell growth was monitored by bioluminescence imaging by luciferin injection, under anesthesia.

Histology and immunohistochemistry: In both models, tumor and organ tissues were analyzed by histology. Tumor specimens were stained for microvessels (anti-CD31 1:240 dilution), HYAL-

4 (anti-HYAL-4 1:1,000 dilution) and Ki67 (anti-Ki67, 1:240 dilution) using an immunohistochemistry procedure described before (16,17,25,32). Microvessel density (MVD)

and proliferation index were determined by counting microvessels and Ki67 positive nuclei, respectively under a Nikon H550L microscope; magnification, 400X.

Statistical analyses: JMP Pro 13 and GraphPad Prism (Version-4.03) software were used for analyses. Differences in the expression of HYAL family transcripts between groups were evaluated by Mann-Whitney U test as data were not normally distributed according to Shapiro-

Wilk test; P-values are two-tailed. Association of V1 expression with clinical and outcome parameters was determined by logistic regression and Cox proportional hazard models. Kaplan-

Meier plots with log-rank statistics were prepared to determine if V1 expression categorized BC patients into risk categories for a specific outcome. Efficacy analyses were based on optimal cut-off values from the ROC curves. Bootstrap modeling was used to calculate mean, median and 95% CI of cut-off values of sensitivity and specificity. Mean ± SD was computed for quantifiable parameters (e.g., cell number, % motility, % invasion, tumor volume). Differences among the transfectants were compared by one-way ANOVA followed by either unpaired t-test;

P values were two-tailed.

RESULTS

HYAL-4 is significantly elevated in BC: We previously established that HYAL-1 expression is significantly elevated in BC tissues and associates with poor clinical outcome (32-34). To investigate whether other HYAL family members were also differentially expressed in BC, we measured HYAL-2, HYAL-3, HYAL-4 and PH20 transcript levels in normal bladder (NBL) and

BC tissues. Since HYAL-P1 is a pseudogene (19), its expression was not examined. Although

HYAL-2 levels were significantly elevated only in high-grade MIBC tissues, the difference between NBL and high-grade MIBC tissues was < 2-fold (Figure 1A). In NBL and BC tissues,

HYAL-3 and PH20 expression was 100 – 1000 fold lower than HYAL-2 and HYAL-4 (Figure 1A).

Compared to NBL tissues, HYAL-4 levels were 2.5 and 24-fold elevated in low- and high-grade

BC tissues, respectively; in high-grade tissues the levels were also 10-fold elevated in MIBC compared to non-MIBC tissues (Figure 1B). The tissue survey panel showed that HYAL-4 expression was significantly elevated in BC (n=20) compared to NBL (n=5) tissues. The levels were also significantly higher in testicular cancer tissues (n=18) compared to the normal testis

(n=7; Figure S1 A).

HYAL-4 variant V1 is generated by insertion of a novel exon”: Since HYAL-4 functions have not been studied in any biological system, we cloned the full length HYAL-4 cDNA from the

HT1376 BC cell line and 15 high-grade BC tissues (Figure S1 B). Sequence analyses revealed that 14 tissues and the cell line expressed a splice variant of HYAL-4, which we labeled “V1”; wild type (Wt) HYAL-4 transcript was detected in only one specimen (Figure S1 B, C). When compared to the Wt sequence, V1 contains a 53 base insertion between exons 4 and 5 (Figure

S1 C). Comparison of the HYAL-4 genomic sequence (GenBank accession # AC006029) and the HYAL-4 Wt mRNA (GenBank accession # NM_012269) to V1 mRNA sequence revealed that the 53 base insertion in V1 maps within intron 4. Therefore, intron 4 contains a previously undescribed exon of 53 nucleotides - exon 4’ (Figure S1 C). Exon-4’ introduces a premature stop codon resulting in a 349 aa truncated protein, of which the first 348 aa are identical to the

Wt sequence (Figure S1 D). Furthermore, HYAL-4 transcript containing exon-4’ is commonly expressed in the bladder tissues. The cloned V1 sequence has been deposited in GenBank

(accession #: BankIt2227219 Seq1 MN902225; BankIt227219 Seq2 MN902226). HYAL-4 Wt protein is GPI-anchored and, based on the prediction, 457Glycine is the possible GPI anchor site

(21). Since V1 lacks the GPI anchor site, it should be secreted (Figure S1 D).

BC tissues mainly express V1: We evaluated expression of Wt and V1 transcripts in bladder tissues by RT-qPCR using Wt and V1 specific primers. While the Wt transcript was almost

undetectable in bladder tissues, V1 levels were 13- and 30-fold higher in high-grade MIBC BC tissues compared to low-grade BC and NBL tissues, respectively (Figure 1C, D). Similarly, V1 levels were 10-fold up-regulated in high-grade MIBC specimens compared to high-grade non-

MIBC tissues (Figure 1D). V1 levels were 3-fold higher in high-grade MIBC tissues from patients who developed metastasis or died from disease during follow-up compared those who did not

(Figure 1E). This suggests that V1 levels likely provide prognostic information beyond MIBC status. V1 levels also risk-stratified patients for metastasis and cancer specific survival (CSS;

Figure 1F, G). In univariate analysis, V1 predicted metastasis and CSS (Tables S4). In order to address whether V1 levels could predict clinical outcome in the presence of other demographic and clinical parameters we performed multivariate analysis that included age, gender, grade, T- stage, N-stage, concomitant CIS, and V1 levels. In this analysis, V1 was an independent prognostic predictor for metastasis and CSS (Table S5). Efficacy analyses revealed that V1 had

~90% sensitivity and specificity to predict metastasis and CSS (Table S6). A custom- synthesized HYAL-4 antibody detected a 40-42 kD protein in aqueous BC tissue extracts and levels were > 5-fold higher in high-grade tumors (Figure 2A, Table S7 (relative densities)). The estimated mol. wt. of the V1 polypeptide (349 aa) is ~ 40 kD. HYAL-4 protein was extracted from tissues in the absence of detergent, suggesting that HYAL-4 protein was secreted in the extracellular milieu and was not membrane-bound.

A recombinant HYAL-4 protein lacking N- and C-termini was reported to have Chase activity (21,22). Therefore, we measured Chase activity in bladder tissue extracts using a novel

ELISA-like Chase activity assay. Chase activity was detectable in bladder tissue extracts and was 10-fold elevated in high-grade BC tissues (Figure 2B). Immunohistochemical localization showed diffuse HYAL-4 staining, consistent with a secreted protein and staining intensity was significantly higher in BC tissues from patients who developed metastasis during follow-up

(Figure 2C; S2 A, B). These results show that V1 expression is up-regulated in high-grade BC tissues, consistent with a corresponding increase in Chase activity.

V1 protein has Chase activity: At present, biological functions of neither Wt nor V1 proteins are known. We stably expressed FLAG-tagged Wt or V1 protein in BC cell lines (T24, 253J and

HT1376). Control transfectants were generated with empty vector (EV). We could only generate

EV and V1 transfectants of an immortalized normal urothelial cell line (Urotsa); several attempts to generate viable Wt transfectants of Urotsa failed. RT-qPCR confirmed expression of Wt and

V1 transcripts in respective transfectants (Figure S3 A). Although two or more protein species were detected by an anti-FLAG antibody, the major bands in Wt and V1 transfectants had a mol. wt. consistent with the expected size of Wt (~50 kD) and V1 (~40 kD) proteins. However, the custom-synthesized HYAL-4 antibody detected only the ~40 kD protein in all transfectants, which was overexpressed in V1 transfectants (Figure 2D). The HYAL-4 antibody also detected the ~40 kD protein in the conditioned medium (CM) of V1 transfectants, demonstrating V1 secretion (Figure 2D). Immunoprecipitation of cell lysates using an anti-FLAG antibody followed by a HYAL-4 antibody from Sigma-Aldrich that is generated against the C-terminal portion (aa

383 – 480) of HYAL-4, detected Wt protein. The antibody could not detect V1 protein because this C-terminal portion is absent in V1 (Figure S3 B). Although the custom-synthesized HYAL-4 antibody should detect both Wt and V1 proteins, it only detected the FLAG-tagged V1 protein immunoprecipitated from the lysates and CM of the V1 transfectants (Figure S3 B). By shRNA transfection, we silenced HYAL-4 expression in HT1376 and T24 cells (Figure 2D, S3 C). Since the HYAL-4 antibody showed knockdown of HYAL-4 protein in shRNA transfectants, it demonstrates the specificity of the antibody which appears to preferentially detect V1 protein

(Figure 2D, Table S7).

Characterization of Chase activity: Measurement of Chase activity showed that only V1 transfectants secrete > 5-fold Chase activity in their CM compared to EV (Figure 2E). Chase activity was not detected in T24 and HT1376 shRNA transfectants (Figure 2E). The pH activity profile of Chase activity in both a high-grade BC tissue extract and HT1376 V1 transfectant CM showed that Chase activity had a pH optimum of 5 – 5.5 (Figure 2F). Chase activity also showed substrate specificity in degrading various types of chondroitin sulfates (CS). Efficiency of Chase activity in HT1376 V1 cell CM and a high-grade BC tissue extract was the following:

CS-Shark/CS-C > CS-D > CS-A (Figure 2G); CS from shark cartilage is mostly (~80%) chondroitin-6-sulfate, the majority of which is CS-C (35-38).

V1 induces a malignant phenotype in bladder cells. Functions of V1 or any eukaryotic

Chase have not been evaluated in any biological system. We examined if V1 or Wt expression affects proliferation, anchorage-independent growth, chemotactic motility, and invasive potential in bladder cells. Growth rate of EV, Wt and V1 transfectants of T24, HT1376 and Urotsa was similar; however, V1 transfectants of 253J cells had a faster growth rate (1.5-fold; P<0.001;

Figure S3 D). When grown on ultra-low attachment plates in stem cell medium, T24-V1 transfectants formed 5-fold more and larger spheroids (Figure 3A). V1 also induced spheroid formation in the immortalized urothelial cell line, Urotsa (Figure 3A). Similarly, V1 transfectants of 253J and HT1376 cells formed 2.5-5-fold more and larger soft agar colonies than EV cells

(Figure 3B). In all cell lines, Wt transfectants consistently and significantly had decreased spheroids and soft agar colonies than EV transfectants (Figure 3A, B). In T24 and HT1376

HYAL-4 shRNA transfectants, spheroid formation was completely inhibited (Figure S3 E).

Increased aldehyde dehydrogenase-1 (ALDH) expression and activity are characteristics of cancer stem cells (CSCs) (39,40). ALDH transcript levels were up-regulated in

V1 transfectants (Figure S3 F, S4 A, B). ALDH activity was also 3-4-fold higher in V1 transfectants when compared to EV and Wt (Figure 3C, S3 F). A stable phenotype we observed is that ~10% of V1-expressing cells float in adherent monolayer cultures. These “V1-floating”

(V1-FL) cells regenerate adherent monolayer cultures in which, again, ~10% of cells float.

These self-renewing V1-FL cells have significantly higher ALDH activity, especially in Urotsa

(Figure 3C; S3 F).

In a wound-healing assay, 253J-V1 transfectants were more motile. While ~100% wound closure was observed in 12 h for V1-expressing cells, only 71% and 60% wound closure was observed for EV and Wt cells at 20 h, respectively (Figure 3D). A similar difference in wound closure was observed for EV and V1 transfectants of Urotsa cells (Figure S4 C). HYAL-4 shRNA transfectants of T24 cells were significantly less motile than control shRNA transfectants. Even at 24 h time point, wound closure was < 50% in HYAL-4 shRNA transfectants as compared to > 80% wound closure in the control transfectants (Figure S3 G). In all cell lines, V1 transfectants showed 1.5-3-fold higher chemotactic motility than EV transfectants (Figure 4A). Similarly, V1 transfectants induced an invasive phenotype even in

Urotsa cells; in all cell lines, V1 transfectants were 2-3-fold more invasive than EV transfectants

(Figure 4A). Contrarily, Wt transfectants showed 2-10-fold lower chemotactic motility and invasive activity compared to EV transfectants (Figure 4A). As expected, both HYAL-4 shRNA transfectants in T24 and HT1376 cells showed 2-8-fold lower chemotactic motility and invasive activity (Figure S4 D).

V1 induces invasive signature in bladder cells. There is consensus that basal phenotype in urothelial cells is characterized by expression of cytokeratin (KRT) 5, 6 and 14 (3,39,40).

Transcript levels of these cytokeratins, as well as, of CD44 and ALDH1 were elevated in V1 transfectants of HT1376 and T24 cells (Figures S4 A, B). V1 did not significantly alter the low

expression of KRT20 or UPK3A (luminal characteristics) in HT1376 cells, but the expression of

UPK3A decreased in V1 transfectants of T24 cells (Figure S4 A, B). Epithelial mesenchymal transition and invasive phenotype markers were also upregulated in V1 transfectants; Vimentin,

β-catenin, N-Cadherin, MMP-9 levels were elevated by 3 - 5-fold (Figure 4B, Table S7).

CD44 is a substrate of V1. Since Chase activity was detectable in the CM of V1 transfectants, we used a monoclonal antibody that specifically detects CS-A and CS-C on CSPGs (anti-CS monoclonal antibody, clone CS-56). While CSPGs were detectable in EV and Wt transfectants, their levels decreased (> 5-fold) in V1 transfectants (Figure 4C, Table S7). Since this antibody specifically detects CS, a decrease in detection of CSPGs suggests loss of CS chains, not necessarily loss of protein due to decreased expression or increased protein degradation. To detect the CSPGs lacking CS chains, we used an anti-chondroitin-6-sulfate monoclonal antibody (MAB2035) that recognizes a neoepitope generated after chondroitin-6-sulfate chains are removed from a CSPG. Levels of a ~70 kD protein bearing this neoepitope (referred to as stub) were highly elevated in both cells and CM of V1 transfectants from all cell lines (Figure

4C). The mol. wt. of this neoepitope-containing protein (called “stub”) was similar to the reported mol. wt. of soluble CD44 (sCD44). sCD44 has been shown to be up-regulated in cancer (41,42).

Although CD44 mRNA expression was 2-3-fold elevated in V1 transfectants (Figure S4

A, B), CD44 protein levels were not consistently up- or down-regulated in these cells. For example, V1 transfectants of T24, HT1376 and Urotsa cells more often showed a decrease in

CD44 protein; however, an increase in CD44 protein was observed in 253J V1 transfectants

(Figure 4C). Regardless of the cell-associated CD44 protein levels, while the levels of sCD44 (~

70 kD) were nearly undetectable in the CM of EV and Wt transfectants of all cell lines, sCD44 levels were increased 10 - 168-fold in V1 transfectants (Figure 4C, Table S7).

Immunoprecipitation of stub from the CM of HT1376 V1 transfectants followed by

immunoblotting with an anti-CD44 antibody revealed that the ~70 kD stub is sCD44 (Figure 4D).

Since sodium dodecyl sulfate was used during immunoprecipitation (Methods section), it establishes that stub is sCD44 and not another protein complexed and co-immunoprecipitated with sCD44. Downregulation of CD44 in HT1376 V1 transfectants by CD44 siRNA not only decreased CD44 levels in cells and CM, but also decreased stub levels (Figure 4E, Table S7).

This further confirmed that in bladder cells, CD44 is the primary substrate of V1’s Chase activity, and that V1’s Chase activity is involved in the generation and secretion of sCD44.

Downregulation of CD44 in HT1376 V1 transfectants decreased spheroid formation by 3-fold, and spheroids were smaller (Figure 4F, Figure S4 E). Motility and invasion of V1 cells transfected with CD44 siRNA were also inhibited by 2-fold and 5-fold, respectively, and resembled the HT1376 EV transfectants (Figure 4A, G).

V1 induces tumorigenesis in non-tumorigenic Urotsa cells. Since V1 expression induced a stem cell-like and invasive phenotype, we examined whether V1 transfectants of Urotsa cells could form tumors in NOD-scid IL2Rgnull mice. As expected, Urotsa EV cells did not grow beyond 100 mm3 (Figure 5A-a, b, c). Furthermore, histology showed that the mass was keratinized and mostly devoid of cells (Figure 5A-d). Contrarily, Urotsa V1 cells formed large angiogenic tumors that invaded skeletal muscle and subcutaneous fat layer (Figure 5A-a, b, c, d). V1 tumors expressed HYAL-4, and had high microvessel density and proliferation index

(positive Ki67 nuclei; Figure 5A-d, Figure S4 F). The keratinized mass of EV cells was devoid of microvessels and Ki67 staining, and did not express HYAL-4 (Figure 5A-d, Figure S4 F).

V1 promotes tumor growth at low cell density. Since V1 expression induced tumorigenesis in non-tumorigenic Urotsa cells, we evaluated if V1 transfectants could form tumors at a low cell density compared to EV transfectants. In the T24 subcutaneous model, we implanted 1,500 EV or V1-FL cells. Growth of V1 tumors at low density was 6-fold higher than EV tumors (Figure

5B-a, b, c). While the small EV tumors were not invasive, V1 tumors invaded skeletal muscle, blood vessels and subcutaneous fat layer (Figure 5B-d). V1 tumors expressed HYAL-4 and had higher microvessel density and proliferation index than EV tumors (Figure 5B-d, Figure S4 G).

V1 promotes tumor growth and metastasis in HT1376 model. We first compared the tumor growth of EV, Wt and V1 transfectants in the HT1376 subcutaneous model. Tumor growth rates were 3- and 5-fold higher for V1 cells than EV and Wt (Figure 6A-a, b, S4 H). Compared to EV tumors, Wt tumors were indolent and V1 tumors invaded muscle and subcutaneous fat layer

(Figure 6A-c).

In the orthotopic intravesical implantation model, bioluminescence imaging detected bladder tumors in 3/4 animals implanted with V1 cells by 19 to 23 weeks (Figure 6B-a). While only 1/4 animals implanted with EV cells developed a detectable signal at 32 weeks

(experimental endpoint), none of the animals implanted with Wt cells developed tumors (Figure

6B-a). At 19 – 23 weeks (end point for V1 animals) there was visual metastasis to lung and spleen (Figure 6B-b). No such lesions were observed in animals implanted with EV or Wt cells.

Histologically, V1 tumors invaded muscle, perivesicle fat and blood vessels; however, no visible tumor growth was observed in EV and Wt bladders (Figure 6B-c). Histology confirmed metastasis to lung and spleen in the animals implanted with V1 cells, whereas metastasis was not observed in animals implanted with EV or Wt cells (Figure 6B-c). These results establish the high tumorigenic and metastatic potential of V1 cells as compared to EV and, notably, Wt cells.

DISCUSSION

This is the first study that identifies and characterizes a naturally occurring eukaryotic

Chase. Moreover, thes study demonstrates that this Chase functions as a molecular driver of

BC and potential predictor of clinical outcome. The urothelium of the bladder is protected from

noxious substances in urine by a glycosaminoglycan layer. While urothelial glycosaminoglycans exist as free chains, several heparan sulfate proteoglycans and CSPGs have been shown to positively or negatively regulate BC progression (4,43). Glycosaminoglycan-degrading enzymes, such as heparanase and from our work, hyaluronidase, also promote BC progression

(4). This study for the first time links HYAL4-V1 and its Chase activity to biological systems in general, and to cancer biology specifically. Salient features of this study are the following: 1.

Splice variant V1 of HYAL-4 gene is the first example of a naturally-occurring secreted Chase and it is expressed in bladder cancer cells and patients’ tumors. 2. A novel high-throughput assay for measuring Chase activity in patients’ tissues and cells. 3. V1 is a potential predictor of metastasis and cancer-specific survival. 4. Expression of V1 in normal urothelial cells induces/promotes tumorigenesis and an invasive, cancer stem cell phenotype. 5. Stem cell marker/driver CD44 is the primary substrate of V1 in bladder cells and mediates its malignant functions. Removal of chondroitin sulfate from CD44 by V1 generates soluble CD44 (sCD44), an established marker in several cancers. 6. In several preclinical models, including an orthotopic model, V1 induces tumorigenesis, tumor growth at low density, muscle and vascular invasion, angiogenesis and metastasis.

Two decades ago, Stern’s group proposed that HYAL-4 is a paralog of hyaluronidases and degrades CS (19). A decade later Kaneiwa et al identified a chondroitin hydrolase from

Caenorhabditis elegans and also showed that HYAL-4, lacking both N- and C-terminal sequences, has Chase activity (21,22,44). In our study, except for the C-terminus FLAG-tag, intact Wt (481 aa) and V1 (349 aa) proteins were expressed in bladder cells. V1-Chase is active at pH 5 – 6 (optimum 5.5), cleaves CS-chains from CD44 in situ, and is detected in “non- detergent” bladder tumor extracts. This suggests that a functionally active Chase is present in the microenvironment of invasive bladder tumors. Therefore, CSPG functions are likely

regulated by a dynamic process of CS addition by CS-synthases and CS removal by a soluble

Chase such as, V1.

Although V1 protein lacks 132 C-terminal aa, it is enzymatically active. Mutational analysis has shown that 131Glu, 205Tyr, aa residues 261 to 265, and 305Gln are essential for the

Chase activity of HYAL-4 (21). All of these residues are present in V1. Since Wt protein lacks detectable Chase activity, it suggests that either the 132 aa region or the anchoring of Wt protein on the plasma membrane inhibits Chase activity. Our novel Chase activity ELISA-like assay showed that CS-C and CS-D were preferred substrates of V1. Kaneiwa et al used a different assay and found similar substrate specificities for HYAL-4 lacking the N- and C- terminal aa (21,22,44).

HYAL-4 is the second member of the HYAL family that is up-regulated in high- grade/MIBC specimens and shows potential for predicting metastasis and CSS. In the tissue survey panel, in addition to BC, testicular cancer also showed HYAL-4 up-regulation. It is possible that like BC, testicular tumor tissues exclusively express V1. Demonstration of the

Chase activity of V1 should allow investigation into the biological functions of Chase/HYAL-4 in normal physiology.

Data presented in this study show that V1-Chase drives malignant functions in urothelial and BC cells. V1-expressing immortalized urothelial and BC cells acquired and/or had enhanced anchorage-independent growth, CSC-like properties, chemotactic motility, and invasive activity. V1-induced invasive phenotype was consistent with an upregulation of MMP-9,

β-catenin, Vimentin and N-Cadherin. V1-expressing normally non-tumorigenic immortalized urothelial cells, Urotsa, formed angiogenic and invasive tumors. V1-expressing cells formed muscle invasive and spontaneously metastatic tumors at low density. The indolent phenotype of

Wt cells in all assays was similar to HYAL-4 silencing in BC cells. This raises a possibility that the membrane-bound HYAL-4 protein negatively regulates tumor growth and progression, which may explain the almost undetectable expression of Wt in both normal bladder and BC specimens.

In this study, we could not examine the function of V1 in BC progression in an immunocompetent mouse model. This is because the mouse version of V1 is unknown, and may not even exist as V1 is generated by alternative splicing of the HYAL-4 gene in human bladder tumors. Without knowing the nucleotide sequence and the splice junctions within the mouse HYAL-4 gene, it is not possible to artificially “create” a mouse version of V1, which could then be utilized to transform mouse bladder tumor cell lines (e.g., MBT-2, MB49). Based on these first in vivo observations of human V1 function, future studies aimed at identifying a potential mouse Chase expressed in bladder should aid in determining how V1 drives BC growth and progression in an immunocompetent environment.

Bladder urothelium is renewed every six to twelve months and consists of umbrella, intermediate and basal cell layers. About 10% of the basal urothelial cells are stem cells that proliferate and differentiate to maintain urothelial integrity (45). Although not well characterized,

BC stem cells share some pattern of cytokeratin expression with urothelial basal cells, i.e., high expression of KRT5, KRT14 and low expression of KRT20. BC stem cells are also CD44+,

ALDH1+ and show anchorage independence and tumor generation (39,40). V1-expressing cells have increased expression of basal cytokeratins (KRT5; KRT6; KRT14), CD44, and ALDH1, as well as, ALDH activity. V1-expressing cells also possess self-renewal properties and demonstrate a malignant phenotype in four in vivo models, including tumor formation in the non- tumorigenic Urotsa cells. These molecular and phenotypic attributes show it is likely that V1 induces urothelial stemness.

Data generated with monoclonal antibodies against CD44, CS-C chains present on

CSPGs, and stub confirm that CD44 is the primary substrate of V1-Chase. Downregulation of

CD44 attenuated the malignant phenotype of V1-expressing cells. CD44, a CSPG, displays chondroitin-6-sulfate chains. CHST3 and CHST7, which transfer a sulfate group onto the N- acetyl-D-galactosamine residues in CSPGs, are known to post-translationally modify CD44

(7,8,10,46). The two CS attachment sites (180S; 190S) map within the stem region that is common in all CD44 isoforms (47). CS modification of CD44 negatively regulates hyaluronic acid binding to CD44, but increases it’s binding to fibrinogen and collagen matrices (8,9,11). The extracellular domain of CD44 is shed in the extracellular matrix (sCD44) following cleavage of

CD44 by ADAM10 at 249S (48-50). Although sCD44 is well studied and prevalent in many tumors, the function of sCD44 or the remaining intracellular domain is largely unknown. V1’s

Chase activity not only removes CS chains from CD44, but also promotes CD44 shedding and induces a malignant phenotype in bladder cells. Therefore, it is possible that CS attachments on

180S and 190S mask the ADAM10 cleavage site at 249S and removal of CS by V1-Chase exposes this site, allowing cleavage and release of the stub-containing sCD44 into the medium (Figure

6C). This is supported by the observation that stub is more abundant in the CM compared to the cell-associated stub. This adds an additional layer of complexity to the regulation of CD44 functions in cancer cells (Figure 6C).

While the functions of CSPGs, and to some degree chondroitin synthases, are well studied in normal and cancer cells, this is the first study on a naturally-occurring eukaryotic

Chase. This study also connects the Chase to human disease in general, and in particular, to cancer biology. We demonstrate that V1-Chase induces a malignant phenotype in bladder cells by removing CS chains from CD44 and increasing its shedding. Furthermore, V1 is a potential

predictor of clinical outcome in BC patients. Finally, as a driver of cancer growth and malignant progression, V1-Chase could be targeted for developing new treatments for cancer.

Acknowledgements: This study is dedicated to the memory of Ms. Neetika Dhir who, together with Dr. Vinata Lokeshwar, first cloned HYAL-4 V1. Ms. Dhir passed away too soon on

December 22, 2008. The research reported in this publication was partly supported by the

National Cancer Institute of the National Institutes of Health, under the awards 1R01CA227277-

01A1 (VBL), 1F31 CA236437-01 (DSM) and 1F31CA210612-01 to ARJ) and from the United

States Army Medical Research and Development Command (USAMRDC) of the Department of

Defense, under award number W81XWH-18-1-0277 (VBL). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the

National Institutes of Health.

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FIGURE LEGENDS

Figure 1: Expression of hyaluronidase gene family in tissues. A - D: Transcript levels of hyaluronidase gene family members in bladder tissues. Normal bladder: NBL (n=31); low-grade

BC: LG (n=7); high-grade non-muscle invasive BC: HG NMIBC (n=6); HG MIBC (n=39). (A):

HYAL-2, HYAL-3, PH20; (B): HYAL-4 (C): HYAL-4 Wt; (D): V1. E: V1 levels of HG MIBC specimens further stratified by clinical outcome (metastasis and CSS). Data: Mean ± SEM. P- values two-tailed; Mann-Whitney U test. F and G: Kaplan-Meier plot showing risk-stratification of the cohort by V1 mRNA levels for metastasis and CSS.

Figure 2: HYAL-4 expression and Chase activity in bladder tissues and bladder cell transfectants. A: Immunoblot analysis of bladder tissue extracts using anti-HYAL-4 antibody.

B: Chase levels (U/mg protein) in bladder tissue extracts measured by Chase assay. C: HYAL-

4 staining in representative NBL and BC tissues. Magnification: 400X. See Figure S2 for enlarged photos and quantification. D: Immunoblot anlaysis of cell lysates and CM of EV, Wt, and V1, Ctr, and HYAL-4 shRNA transfectants for indicated proteins; loading control: Actin.

HYAL-4 peptide block: the peptide was added during incubation with the anti-HYAL-4 antibody.

Note for A and D: In each blot, all sample lanes were run on the same gel with the same exposure time; a gap denotes those samples that were not contiguous within the gel. E: Chase activity (U) was measured in the CM of bladder cell transfectants (left panel) and in the CM of control (Ctr) and HYAL-4 shRNA transfectants (right panel). The activity was normalized to cell number. Data: Mean ± SEM; quadruplicate. F: Normalized Chase activity in the CM of HT1376 transfectants and in a high-grade bladder tumor tissue extract (HG-TBL) at indicated pH. Data:

Mean ± SEM; quadruplicate. G: Chase activity in the CM of HT1376 V1 transfectants and a

HG-TBL extract measured against different chondriotin sulfate substrates. Data: Mean ± SEM; quadruplicate.

Figure 3: Phenotypic characterization of bladder cell transfectants. A and B: Anchorage- independent growth. (A): Growth of bladder cell transfectants in Spheroids at day-7 or (B): soft- agar at day-21. Right panels: quantification of spheroid and soft agar colonies. Magnification:

100X. Data: Mean ± SEM; n = 5 – 10 fields. C: Measurement of ALDH activity by flow- cytometry. Right panel: Mean fluorescence intensity quantification. Data: Mean ± SD. D: Motility of 253J transfectants measured in a scratch-would assay. Data: Mean ± SD; quadruplicate.

Figure 4: Phenotypic characterization of bladder cell transfectants and CD44 silencing.

A: Chemotactic motility and invasive activity of EV, Wt and V1 transfectants of bladder cells at

18 and 48 hours, respectively. Data: Mean ± SEM; triplicate. B and C: Immunoblotting of bladder cell transfectants for indicated proteins; loading control: Actin. Note: In each blot, all sample lanes were run on the same gel with the same exposure time; a gap denotes those samples that were not contiguous within the gel. D. Immunoprecipitation of HT1376 V1 CM by anti-Stub or control (mouse) antibody followed by immunoblotting with anti-CD44 antibody.

Input: CM without immunoprecipitation. Note: HC (heavy chain) and LC (light chain) of the IgG present in the control and Stub immunoprecipitates. E – G: Analyses of CD44 siRNA transient transfectants of HT1376 V1 cells. (E): Immunoblotting for indicated proteins; (F): Spheroid growth; quantification of the data is presented in Figure S4 E. (G): Percent chemotactic motility and invasion (Mean ± SD; triplicate).

Figure 5: Tumor growth characteristics of HYAL-4 transfectants in Urotsa and T24 xenograft. A: Urotsa subcutaneous xenograft in NOD/SCID mice (n=5/transfectant). (a): Tumor volume. (b): Tumor (V1) weight or weight of the keratinized mass (EV) at necropsy. Data: Mean

± SEM. (c): Tumors (V1) and keratinized mass at the injection site (EV). (d): Histology and IHC for indicated proteins. Arrows show pyknotic tumor cells/nuclei present in the keratinized mass

of EV xenograft and tumor invading the skeletal muscle in V1 xenograft. B: T24 subcutaneous xenograft in athymic mice (n=5/transfectant). (a): Tumor volume. (b): tumor weight. Data: Mean

± SEM. (c): tumors at necropsy. (d): Histology and IHC for indicated proteins. Arrows show V1 tumor cells invading into skeletal muscle and a microvessel. Magnification for all photos: 400X.

Figure 6: Tumor growth and metastasis characteristics of HYAL-4 transfectants in

HT1376 xenograft. A: HT1376 subcutaneous xenograft in athymic mice (n=5/transfectant). (a):

Tumor volume. Data: Mean ± SEM. (b): tumors at necropsy. (c): Histology of tumors. Note:

Mostly keratinized mass is present in Wt xenograft. In V1 xenograft, tumor is invading the muscle and subcuatenous fat layer. B: Orthotopic implantation of luciferase expressing HT1376 transfectants in the bladders of athymic mice (n=4); Bioluminescence imaging at indicated weeks. (a): Tumor imaging. (b): Organ metastasis detected at necrospy (19th week) in the animal implanted with V1 cells. (c): Histology of indicated organs. Magnification: 400X. C:

Proposed model of V1-CD44-ADAM10 interaction. Upregulation of V1 increases Chase activity in the extracellular milieu of tumor cells. Secreted V1-Chase degrades CS chains attached to the stem-region in the extracellular domain of CD44 generating the neoepitope “stub”. The removal of CS chains exposes the ADAM10 cleavage site, causing CD44 cleavage and the release of soluble CD44 (sCD44).

A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Bladder Cancer Patients.

Vinata B. Lokeshwar, Daley S Morera, Sarrah L Hasanali, et al.

Clin Cancer Res Published OnlineFirst February 24, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-2912

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/02/22/1078-0432.CCR-19-2912.DC1

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