CDCP1 expression is frequently increased in aggressive urothelial carcinoma and promotes urothelial tumor progression

The prognosis of patients with advanced urothelial carcinoma (UC) remains poor and improving treatment continues to be a major medical need. CUB domain containing protein 1 (CDCP1) is a known oncogene in various types of solid cancers and its overexpression is associated with impaired prognosis. However, its role in UC remains undetermined. Here we assessed the clinical relevance of CDCP1 in two cohorts of UC at different stages of the disease. Immunohistochemistry showed that CDCP1 is highly expressed in advanced UC, which significantly correlates with shorter overall survival. Importantly, the basal/squamous UC subtype showed significantly enriched CDCP1 at the mRNA and protein levels. The functional role of CDCP1 overexpression was assessed taking advantage of ex vivo organoids derived from the CDCP1pcLSL/+ transgenic mouse model. Furthermore, CDCP1 knockout UC cell lines were generated using CRISPR/Cas9 technology. Interestingly, CDCP1 overexpression significantly induced the activation of MAPK/ERK pathways in ex vivo organoids and increased their proliferation. Similarly, CDCP1 knockout in UC cell lines reduced their proliferation and migration, concomitant with MAPK/ERK pathway activity reduction. Our results highlight the relevance of CDCP1 in advanced UC and demonstrate its oncogenic role, suggesting that targeting CDCP1 could be a rational therapeutic strategy for the treatment of advanced UC.


Results
UC exhibits an elevated expression of CDCP1, which correlates with shorter overall survival in UC patients. To assess the clinical relevance of CDCP1 in UC, we first performed immunohistochemistry (IHC) staining of human CDCP1 on paraffin-embedded human bladder cancer samples based on the previously described protocol 12 . We examined a cohort of 147 specimens spanning from T1 to T4 stages (Table 1) 29,30 and stratified samples based on their membrane staining for CDCP1 into four groups (negative, weak, moderate and strong). All groups with negative or weak staining intensity were classified as CDCP1-low, while moderate and strong groups were classified as CDCP1-high (Fig. 1A). In line with previous results 28,31 , CDCP1 expression was negatively or weakly expressed in normal urothelium or normal adjacent tissue (NAT) (Supplementary Fig. 1). A large portion of urothelial tumors at T1 and T2 stages were classified as negative/weak for CDCP1 (CDCP1low), while the 35% of T3 and 50% of T4 showed high expression levels of CDCP1 (CDCP1-high) (Fig. 1B). Of note, levels of CDCP1 in muscular invasive bladder cancer (MIBC) stages (T2-T4) are significantly higher than in the non-invasive stage (T1) (Fig. 1C). Most importantly, patients with CDCP1-high expression showed a significant poorer outcome compared to patients with CDCP1-low expression levels (Log Rank mantel cox test P < 0.0001) (Fig. 1D). In parallel, we performed western blot and real-time PCR (RT-PCR) analyses (n = 10 and n = 107 respectively) on additional UC frozen samples and the corresponding NAT. These analyses demonstrated a robust increase of CDCP1 expression at protein and RNA levels in UC when compared to NAT (Fig. 1E,F). Taken together, these data showed that CDCP1 is overexpressed in UC compared to normal tissues, and its overexpression is associated with advanced UC and shorter overall survival (OS).

CDCP1 expression is elevated in advanced UC and enriched in Ba/Sq subtype. To gain further
insights about the expression of CDCP1 in UC, we analyzed CDCP1 expression in two publically available data sets. The first, consisting of 405 tissue specimens of bladder cancer from the cancer genome atlas (TCGA), was classified according to the consensus subtyping approach of muscle-invasive urothelial bladder cancer (MIBC) 32 . In line with our previous results, we observed elevated levels of CDCP1 in UC compared to its NAT ( Fig. 2A). Importantly, we found that CDCP1 levels were strongly associated with the Ba/Sq subtype (Fig. 2B). This was confirmed using a second data set, a single-cell-RNA-sequencing (scRNAseq) of human UC samples 33 . Indeed, analysis of this dataset for cells annotated as epithelial origin revealed that expression of CDCP1 and that of markers reported to be enriched in the Ba/Sq subtype, such as EGFR, KRT5 (CK5) and KRT14 (CK14), have a high degree of co-localization (Fig. 2C). Thereafter, we performed IHC of CDCP1 on a well-established prospectively recruited consecutive cohort of MIBC patients treated with radical cystectomy and adjuvant chemotherapy as previously described (Table 2) 32 . The multivariate analysis on this cohort demonstrated that CDCP1 is not an independent variable for the prediction of patients´ prognosis (Table 3). However, out of 184 analyzed tumors 56% were positive for CDCP1 (Fig. 2D) and we found a strong association between CDCP1 protein expression and the Ba/Sq subtype (Fig. 2E). This association with the Ba/Sq subtype of UC was further validated using the Ba/Sq subtype markers CK5 and CK14 32 (Fig. 2F). Of note, the mRNA levels of CDCP1 reflected the protein levels observed in this cohort (Fig. 2G) and were enriched in the Ba/Sq subtype (Kruskal Wallis: P < 000.1) (Fig. 2H). Together, these results confirm the association of CDCP1 expression with the aggressive Ba/Sq UC subtype. Moreover, considering that several studies have already reported the association of CDCP1 with therapy resistance 6 , we questioned whether this protein has the same impact on MIBC. The OS of chemotreated patients expressing high CDCP1 levels is clearly reduced (Fig. 2I), suggesting that CDCP1 may be a suitable marker for chemotherapy sensitivity and targeting CDCP1 could serve as a novel therapeutic strategy to treat resistant patients.

Transgenic overexpression of CDCP1 induces proliferation in ex vivo mouse organoids and its knockout inhibits proliferation and migration of UC cells.
To model the effect of CDCP1 overexpression in UC, we exploited the previously generated transgenic mouse model for CDCP1 (CDCP1 pcLSL/+ ) and established an ex vivo 3D organoids system that overexpresses CDCP1 12 . Briefly, the bladder of 8 weeks old male CDCP1 pcLSL/+ mice was excised and dissociated into single cells, as previously described 33 . Bladder cells were infected with an adeno-Cre virus to induce the expression of CDCP1 and seeded in Matrigel (Fig. 3A). We examined the effect of CDCP1 expression on the growth of bladder mouse organoids over two weeks. CDCP1 overexpression resulted in the formation of larger and morphologically distinctive organoids compared to the controls (Fig. 3B). Quantification of the organoids area revealed a significant increase in their size when CDCP1 is overexpressed compared to the controls (Fig. 3C). At the molecular level, IHC analysis on mouse organoids confirmed the expression of CDCP1 (Fig. 3D). Importantly, CDCP1 overexpressing organoids expressed high levels of Ki67 and pERK (Fig. 3D). To further assess the relevance of these findings in human UC, we aimed to perform CDCP1 knockout (KO) in CDCP1 expressing cells with the CRISPR/Cas9 method. Firstly, we screened several UC cell lines for their CDCP1 expression. Western blot analysis indicated variable expression of CDCP1 among the tested UC cell lines (Supplementary Fig. 2A). Given the fact that CDCP1 expression was associated with Ba/Sq subtype and it is highly expressed in the Ba/Sq SCaBER cells, we generated KO of CDCP1 in this cell line (Fig. 3E). Interestingly, loss of CDCP1 in SCaBER cells reduced pAKT, pMEK and pERK1/2 levels ( Fig. 2B). Functional analyses showed that CDCP1 depletion reduced both 2D and 3D proliferation (Fig. 3F,G) and migration in this cell line (Fig. 3H). Interestingly, T24 and TCCSUP cells, which are considered non-type 34,35 , showed a similar behavior to SCaBER when knocked out for CDCP1, with the exception for the 2D proliferation ( Supplementary Fig. 2C-F). Collectively, these results demonstrate that CDCP1 expression promotes UC proliferation, while its downregulation reduces proliferation and migration of UC cell lines.

Discussion
Despite the preponderance of studies identifying CDCP1 as a key contributor to oncogenic events in several cancers 6 , the functional role of CDCP1 and its clinical relevance in UC remains poorly characterized. Only two previous studies from the same laboratory indicate its involvement in BCa 28, 31 . Yang et al. showed that N6-methyladenosine modified the mRNA levels of CDCP1 in response to chemical carcinogens, which promoted CDCP1 translation. The same group also showed that CDCP1 is moderately or highly expressed in most of the BCa samples (n = 33) when compared to para-tumor controls and correlated CDCP1 expression with BCa progression 31 . In line with these results, the first part of our study demonstrated that CDCP1 is overexpressed in UC when compared to NAT and is associated with MIBC. Moreover, the impact of elevated CDCP1 expression in UC was investigated by Kaplan-Meier analysis, which indicated that patients expressing high levels of CDCP1 have significantly poorer OS (Fig. 1D).
In the second part of this study, we further explored the clinical relevance of CDCP1 in UC. Indeed, our results obtained from three different data sets, showed significant enrichment of CDCP1 expression in the Ba/ Sq subtype (Fig. 2B,C,E,H). Since CDCP1 is reported to interact with the epidermal growth factor receptor (EGFR) 36 , whose activity is also associated with Ba/Sq tumors in BCa 37, 38 , and a cross-talk between these two proteins is already described in other tumor types [39][40][41] , further studies investigating the potential CDCP1/EGFR cross-talk are needed. Such studies may show that a combinational therapy targeting CDCP1 and EGFR may be effective for the treatment of Ba/Sq bladder tumors.
Another important clinical aspect presented by our study is the association of CDCP1 with therapy resistance, which has been reported in breast and ovarian cancers 6,11,18 . Indeed, the OS of chemotherapy-treated patients expressing high CDCP1 levels was clearly reduced, supporting the hypothesis that CDCP1 is involved in resistance to first-line chemotherapy in UC.
To determine the oncogenic role of CDCP1 in UC, we took advantage of the previously generated mouse model for CDCP1 overexpression in a Cre-dependent manner 12 . Remarkably, CDCP1 overexpression in bladder organoids obtained from this mouse model resulted in larger and well-defined organoids compared to the control groups ( Fig. 3B,C). These preliminary results suggest that CDCP1 overexpression may support oncogenesis in bladder urothelium. Additionally, these results encourage the development of a bladder-specific CDCP1-overexpressing transgenic mouse model to further analyze the oncogenic potential of CDCP1 in UC.     www.nature.com/scientificreports/ At the molecular level, we showed that CDCP1 depletion reduced MEK, ERK and AKT phosphorylation in western blot analyses on SCaBER cells knocked out for CDCP1. The reliance of MAPK/ERK and AKT pathways activation on CDCP1 expression in SCaBER suggests that this protein may play a crucial role in UC progression. Indeed, it was previously reported that the activation of MAPK/ERK and AKT pathways increased tumor growth and cancer cells motility 8,12 . Functional analysis performed with SCaBER cells showed that CDCP1 depletion reduced their 2D and 3D proliferation and migration (Fig. 3F,G,H). Altogether, these findings demonstrate that the growth and migration abilities of SCaBER cells rely on CDCP1 expression, suggesting that Ba/Sq UC patients could benefit from CDCP1-targeting therapies. Therefore, further studies validating the efficacy of CDCP1 inhibition are clearly encouraged.

Materials and methods
Patient cohort and case report. The study was approved by the ethical review board of the Medical Faculty of the University of Bonn (approval number: 036/08 and 093/12) and the Friedrich-Alexander-University Erlangen-Nürnberg (approval number: 329_16B and 97_18Bc). The study was performed in accordance with the Declaration of Helsinki. The study participants were anonymized before their specimens were included in the study cohort. Informed consent was obtained for all the participants. In the first TMA, CDCP1 expression was assessed from patient samples obtained via radical cystectomy or transurethral resection and provided by the University Hospital of Bonn, including benign bladder urothelium and BCa with different stages of disease (T1-T4) as previously described (Table 1) 29,30 . In the second TMA, CDCP1 expression was assessed in a wellcharacterized prospective homogenous MIBC cohort treated by radical cystectomy in conjunction with bilateral lymphadenectomy in curative intent at the Department of Urology of the University Hospital of Erlangen 32 .  11,12,17,21 . CDCP1 staining was evaluated by two experienced pathologists (OH and GK) and only specific membrane expression of CDCP1 was assessed. Staining intensity was classified as negative (0), weak (1), moderate (2) and strong (3). Negative and weak specimens were considered CDCP1-low, whereas moderate and strong staining were considered CDCP1-high. scRNAseq analysis. Data was downloaded as from Chen et al., 2020 33 , using their interactive Shiny R interface. Analysis and dataset processing was performed using Seurat version 4.1.1 running on a mac OS version 12.2.1 (Monterey). Analysis was performed using standard Seurat dataset processing pipeline. In brief, data was subsetted to include only cells annotated as "epithelial". Data was visualised using the Nebulosa (version 1.4.0) and scCustomize (version 0.7.0) packages ( Table 2). The colour-blind friendly, perceptually uniform and ordered "batlow" colour pallet was used via the R package scico (version 1.3.0). The figure was layed out using Adobe Illustrator version 24.1. Data availability: Publically available scRNAseq data was obtained from chen et al., 2020. Code availability: Code to reproduce scRNAseq data can be found at https:// github. com/ Eomes oderm in.
Cell culture and functional assays. The human UC cell lines used were purchased from ATCC (ATCC, Manassas, VA, US). Cells were cultured in RPMI1640 (Thermo Fisher Scientific) supplemented with 10% FCS (Thermo Fisher Scientific), 1% streptomycin/penicillin (10.000 units/ml Penicillin and 10.000 µg/ml Streptomycin; Thermo Fisher Scientific) and 1% l-glutamine (200 mM; Thermo Fisher Scientific). For the spheroids formation assay, cells were grown in 1.2% Methylcellulose (Sigma-Aldrich) at a density of 25 × 10 3 cells/mL as hanging drops and incubated under standard culture conditions for 72 h. The diameter of the formed spheroids was measured with Image J and spheroids volume was calculated with the formula V = 4/3 × π × r 3 . For the migration assay we put 20 × 10 3 cells on 24-well plate inserts and incubate for 12 h. We then fixed the cells with PFA 4%, performed crystal violet assay and swapped the internal part of the insert´s membrane to eliminate cells which didn´t migrate. We finally counted the migrated cells on the external part of the membrane. For the establishment of the ex vivo organoids model, single cells from the bladder of male CDCP1 pcLSL/+ mice were isolated as previously described 33 . Single cells were suspended (10 5 cells/mL) in DMEM, 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin and infected with adenoviruses (rAAV2/1-CMV-GFP and rAAV2/1-CBA-Cre) via spinoculation at 600*g, 1 h, 32 °C. Cells were then incubated for 1 h at 37 °C and 5% CO 2 . 10 6 cells were suspended in 1 mL of Matrigel (Corning, New York, United States) and 40 µL drops were formed in pre-warmed flat bottom 24-well plates. Matrigel drops were left 30 min to solidify at 37 °C and 5% CO 2 inverting the 24-well     . The doublestranded oligonucleotide was obtained by annealing the following single-stranded oligonucleotides: hCDCP1_ KO_BS, 5′-AAACccgtggtcaggatcggaac-3′; hCDCP1_KO_TS, 5′-CACCgttccgatcctgaccacgg-3′. After the ligation, px459-CDCP1 −/-sgRNA plasmid was transfected in the cells using Lipofectamine™ 3000 (Invitrogen, Thermo Fisher Scientific). After 2 days, the transfected clones were selected with a 4-days Puromycin treatment (0.6 μg/ mL) and expanded with a polyclonal approach to obtain stable cell lines.

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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