Mouse Models of Muscle-invasive Bladder Cancer: Key Considerations for Clinical Translation Based on Molecular Subtypes.

CONTEXT: In the past few years, research has suggested that molecular subtypes in muscle-invasive bladder cancer (MIBC) may be exploited to accelerate developments in clinical disease management and novel therapeutics. OBJECTIVE: To review MIBC mouse models from a molecular subtype perspective, their advantages and limitations, and their applications in translational medicine, based on a PubMed search for publications from January 2000 to February 2018. EVIDENCE ACQUISITION: Publications relevant to MIBC mouse models and their molecular subtypes were identified in a literature review. EVIDENCE SYNTHESIS: We classified the models according to the technique used for their establishment. For xenotransplant and allograft models, the inoculated cells and inoculated locations are the major determinants of molecular subtypes. Although the cell lines used in xenotransplant models can cover most of the basal-squamous and luminal subtypes, allograft models offer a more realistic environment in which to reconstruct aspects of the associated stromal and immune features. Autochthonous models, using genetic and/or chemical stimuli to induce disease progression, can also generate models with basal-squamous and luminal subtypes, but further molecular characterisation is needed since other mutational variants may be introduced in these models. CONCLUSIONS: We identified preclinical MIBC models with different subtype specifications and assessed their promise and current limitations. These models are versatile tools that can reproduce the molecular complexity of MIBC and support novel therapeutic development. PATIENT SUMMARY: Understanding which models of muscle-invasive bladder cancer most accurately represent the clinical situation is important for the development of novel drugs and disease management strategies. We review the different models currently available and their relevance to different clinical subtypes.

TRAIL-mediated apoptosis in breast cancer cells cultured as 3D spheroids.

TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44hiCD24loALDH1hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway. We show that inhibition of the COX-2/PGE2 pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44hiCD24lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance.

1 alpha, 25-dihydroxylvitamin D3 promotes Bacillus Calmette-Guérin immunotherapy of bladder cancer.

Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis(TB), has been used and proven to be one of the most effective treatments for non-muscle invasive bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, thereby limiting the improvement of BCG therapy. Vitamin D deficiency has been associated with a high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy. To test this hypothesis, we examined the impact of 1 alpha, 25-dihydroxyvitamin D3 (1,25-VD) on BCG-induced response in BCa cells and macrophage cells. Our data revealed that 1,25-VD promotes BCG-induced interleukin 8 (IL-8) secretion by BCa cells, consequently inducing the migration of macrophage, THP-1. This THP-1 cell migration promoted by 1,25-VD can be blocked by IL-8 neutralized antibody. Furthermore, 1,25-VD increased BCG-induced expression of macrophage markers in THP-1 cell, and enhanced the BCG-induced THP-1 cytotoxicity against low-grade BCa cells. Importantly, a pre-clinical trial using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model revealed that intravesical co-treatment of 1,25-VD with BCG can prolong mice survival. These data demonstrate a novel mechanism by which 1,25-VD promotes BCG-mediated anti-BCa pathways and provides a platform for improving BCG efficacy with combination of 1,25-VD.

Targeting Underglycosylated MUC1 for the Selective Capture of Highly Metastatic Breast Cancer Cells Under Flow.

The underglycosylated form of the MUC1 glycoprotein, uMUC1, has been identified as a ligand for both E-selectin and ICAM-1 and can play multiple potential roles during rolling and firm adhesion events in the metastatic cascade. Using flow cytometry and confocal microscopy, the T47D and ZR-75-1 cell lines were verified to highly express uMUC1, however it was found that only ZR-75-1 cells expressed the E-selectin binding moiety sialyl Lewis x (sLex). Furthermore, perfusing T47D cells through E-selectin coated microtubes resulted in fast rolling velocities and low numbers of interacting cells and blocking uMUC1 with the SM3 antibody had no effect. ZR-75-1 cells, on the other hand, were highly dependent on the E-selectin:uMUC1 interaction as exemplified by significant increases in cell rolling velocities and decreases in the number of interacting cells when blocking with SM3 or when uMUC1 expression was knocked down via siRNA transfection. Whereas uMUC1 interactions with E-selectin supported cell rolling, P-selectin: uMUC1 interactions exclusively facilitated cell tethering, while L-selectin surfaces supported no cell adhesive interactions. These experimental observations are consistent with molecular dynamics simulations of uMUC1 bound to E-, P-, and L-selectin where the degree of residue contact correlated with the differential adhesion of uMUC1 to each selectin. Finally, an E-selectin and SM3 combined surface coating captured approximately 30% of the total number of interacting cancer cells comparable to the number of adhered cells when utilizing E-selectin and ICAM-1 combined surfaces. The E-selectin/SM3 surface strategy offers a viable method to selectively capture cancer cells from whole blood samples.

Decreased tumorigenesis and mortality from bladder cancer in mice lacking urothelial androgen receptor.

Much fewer mice lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR(-/y)) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR(-/y) mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR(+/y)) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR(-/y) than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR(-/y) mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR(-/y)) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR(+/y)), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.

Phenotypic switch in blood: effects of pro-inflammatory cytokines on breast cancer cell aggregation and adhesion.

Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa) patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLe(x) expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of 'triple-negative' breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments.

Targeting thymic epithelia AR enhances T-cell reconstitution and bone marrow transplant grafting efficacy.

Although thymic involution has been linked to the increased testosterone in males after puberty, its detailed mechanism and clinical application related to T-cell reconstitution in bone marrow transplantation (BMT) remain unclear. By performing studies with reciprocal BMT and cell-specific androgen receptor (AR) knockout mice, we found that AR in thymic epithelial cells, but not thymocytes or fibroblasts, played a more critical role to determine thymic cellularity. Further dissecting the mechanism using cell-specific thymic epithelial cell-AR knockout mice bearing T-cell receptor transgene revealed that elevating thymocyte survival was due to the enhancement of positive selection resulting in increased positively selected T-cells in both male and female mice. Targeting AR, instead of androgens, either via genetic knockout of thymic epithelial AR or using an AR-degradation enhancer (ASC-J9®), led to increased BMT grafting efficacy, which may provide a new therapeutic approach to boost T-cell reconstitution in the future.

Androgen receptor signals regulate UDP-glucuronosyltransferases in the urinary bladder: a potential mechanism of androgen-induced bladder carcinogenesis.

UDP-glucuronosyltransferases (UGTs), major phase II drug metabolism enzymes, play an important role in urinary bladder cancer initiation by detoxifying carcinogens. We aimed to determine if androgens regulate UGT expression via the androgen receptor (AR) pathway in the bladder. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to assess UGT1A levels in the normal urothelium SVHUC cell line stably expressed with AR and in bladder tissues from AR knockout (ARKO) and castrated male mice. Immunohistochemistry was also performed in radical cystectomy specimens. Dihydrotestosterone (DHT) treatment in SVHUC-AR reduced mRNA expression of all the UGT1A subtypes (19-75% decrease), and hydroxyflutamide antagonized the DHT effects. In contrast, DHT showed only marginal effects on UGT1A expression in SVHUC-Vector. Of note were higher expression levels of UGT1As in SVHUC-Vector than in SVHUC-AR. In ARKO mice, all the Ugt1a subtypes were up-regulated, compared to wild-type littermates. In wild-type male mice, castration increased the expression of Ugt1a8, Ugt1a9, and Ugt1a10. Additionally, wild-type female mice had higher levels of Ugt1a than wild-type males. Immunohistochemical studies showed strong (3+) UGT1A staining in 11/24 (46%) cancer tissues, which was significantly lower than in corresponding benign tissues [17/18 (94%) cases (P = 0.0009)]. These results suggest that androgen-mediated AR signals promote bladder carcinogenesis by down-regulating the expression of UGTs in the bladder.

A positive feedback signaling loop between ATM and the vitamin D receptor is critical for cancer chemoprevention by vitamin D.

Both epidemiologic and laboratory studies have shown the chemopreventive effects of 1α,25-dihydroxyvitamin D(3) (1,25-VD) in tumorigenesis. However, understanding of the molecular mechanism by which 1,25-VD prevents tumorigenesis remains incomplete. In this study, we used an established mouse model of chemical carcinogenesis to investigate how 1,25-VD prevents malignant transformation. In this model, 1,25-VD promoted expression of the DNA repair genes RAD50 and ATM, both of which are critical for mediating the signaling responses to DNA damage. Correspondingly, 1,25-VD protected cells from genotoxic stress and growth inhibition by promoting double-strand break DNA repair. Depletion of the vitamin D receptor (VDR) reduced these genoprotective effects and drove malignant transformation that could not be prevented by 1,25-VD, defining an essential role for VDR in mediating the anticancer effects of 1,25-VD. Notably, genotoxic stress activated ATM and VDR through phosphorylation of VDR. Mutations in VDR at putative ATM phosphorylation sites impaired the ability of ATM to enhance VDR transactivation activity, diminishing 1,25-VD-mediated induction of ATM and RAD50 expression. Together, our findings identify a novel vitamin D-mediated chemopreventive mechanism involving a positive feedback loop between the DNA repair proteins ATM and VDR.

Suppression of prostate cancer cell rolling and adhesion to endothelium by 1α,25-dihydroxyvitamin D3.

Adhesion of circulating prostate cancer (PCa) cells to the microvascular endothelium is a critical step during cancer metastasis. To study PCa cell rolling and adhesion behavior, we developed a dynamic flow-based microtube system to mimic the microvascular environment. We found that PCa cell rolling capacity is mediated by E-selectin and can be enhanced by stromal cell-derived factor-1 under different wall shear stresses. Using this device, we tested if the chemopreventive agent, vitamin D, could interfere with PCa cell adhesion. We found that 1α,25-dihydroxyvitamin D(3) (1,25-VD), the bioactive form of vitamin D, reduced PCa cell rolling numbers and increased rolling velocities resulting in a significant decreased number of PCa cells adhering to the microtube. The inhibitory effects of 1,25-VD on PCa cell heterotypic adhesion were further confirmed using microvascular endothelial cells in a static condition. Furthermore, we demonstrated that 1,25-VD can increase E-cadherin expression in PCa cells and promote the homotypic cell-cell aggregation, which can then hinder PCa cell adhesion to the endothelium. Blocking E-cadherin with a neutralizing antibody can reverse 1,25-VD-mediated suppression of PCa cell adhesion to the endothelium. Taken together, our data revealed that 1,25-VD promoted PCa cell aggregation by increasing E-cadherin expression, thus interfering with circulating PCa cell adhesion to microvascular endothelial cells and potentially reducing their metastatic potential.

Down-regulation of NF-kappaB signals is involved in loss of 1alpha,25-dihydroxyvitamin D3 responsiveness.

Vitamin D anti-tumor effect is often found reduced in the late stages of cancer. To uncover vitamin D resistance mechanism, we established a vitamin D-resistant human prostate cancer LNCaP cell line, LNCaP-R, by chronic exposure of cells to 1alpha,25-dihydroxyvitamin D(3) (1,25-VD). The vitamin D receptor (VDR)-mediated transcriptional activity was reduced in LNCaP-R, whereas VDR expression level and DNA-binding capacity were similar compared to parental cells (LNCaP-P). The expressions of the key factors involved in VDR transactivity, including CYP24A1 and VDR-associated proteins are all increased in LNCaP-R cells, and yet treatment with ketoconazole, P450 enzymes inhibitor, as well as trichostatin A (TSA), a histone deacetylase inhibitor, did not sensitize LNCaP-R cells response to vitamin D, suggesting that neither a local 1,25-VD availability, nor VDR-associated proteins are responsible for the vitamin D resistance. Interestingly, nuclear factor-kappaB (NF-kappaB) signaling, which is critical for 1,25-VD/VDR activity was found reduced in LNCaP-R cells, thereby treatment with NF-kappaB activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), can sensitize LNCaP-R vitamin D response. Together, we conclude that NF-kappaB signaling is critical for vitamin D sensitivity, and dysregulation of this pathway would result in vitamin D resistance and disease progression.

Neutropenia with impaired host defense against microbial infection in mice lacking androgen receptor.

Neutrophils, the major phagocytes that form the first line of cell-mediated defense against microbial infection, are produced in the bone marrow and released into the circulation in response to granulocyte-colony stimulating factor (G-CSF). Here, we report that androgen receptor knockout (ARKO) mice are neutropenic and susceptible to acute bacterial infection, whereas castration only results in moderate neutrophil reduction in mice and humans. Androgen supplement can restore neutrophil counts via stabilizing AR in castrated mice, but not in ARKO and testicular feminization mutant (Tfm) mice. Our results show that deletion of the AR gene does not influence myeloid lineage commitment, but significantly reduces the proliferative activity of neutrophil precursors and retards neutrophil maturation. CXCR2-dependent migration is also decreased in ARKO neutrophils as compared with wild-type controls. G-CSF is unable to delay apoptosis in ARKO neutrophils, and ARKO mice show a poor granulopoietic response to exogenous G-CSF injection. In addition, AR can restore G-CSF-dependent granulocytic differentiation upon transduction into ARKO progenitors. We further found that AR augments G-CSF signaling by activating extracellular signal-regulated kinase 1/2 and also by sustaining Stat3 activity via diminishing the inhibitory binding of PIAS3 to Stat3. Collectively, our findings demonstrate an essential role for AR in granulopoiesis and host defense against microbial infection.

Protective role of 1 alpha, 25-dihydroxyvitamin D3 against oxidative stress in nonmalignant human prostate epithelial cells.

Overproduction of reactive oxygen species (ROS), through either endogenous or exogenous sources, could induce DNA damage, and accumulation of DNA damage might lead to multistep carcinogenesis. The antioxidative effects of vitamin D have been suggested by epidemiological and many in vitro and in vivo laboratory studies. While exploring the antioxidative effects of vitamin D in prostate cells, we found that the active form of vitamin D, 1 alpha, 25-dihydroxyvitamin D(3) (1,25-VD), can protect nonmalignant human prostate epithelial cell lines, BPH-1 and RWPE-1, but not malignant human prostate epithelial cells, CWR22R and DU 145, from oxidative stress-induced cell death. Glucose-6-phosphate dehydrogenase (G6PD), a key antioxidant enzyme, was dose- and time-dependently induced by 1,25-VD. Mechanistic studies using chromatin immunoprecipitation (ChIP) assay revealed that a direct repeat-3 (DR3) vitamin D response element located in the first intron of the G6PD genome can be bound by liganded vitamin D receptor, thereby regulating G6PD gene expression. Increasing G6PD activity and glutathione level by 1,25-VD can scavenge cellular ROS. Moreover, the protective effects of 1,25-VD were abolished by dehydroepiandrosterone, a noncompetitive inhibitor of G6PD activity. Together, our results showed that 1,25-VD can protect nonmalignant prostate cells from oxidative stress-induced cell death by elimination of ROS-induced cellular injuries through transcriptional activation of G6PD activity. The antioxidative effect of vitamin D strengthens its roles in cancer chemoprevention and adds to a growing list of beneficial effects of vitamin D against cancer.