E9 Cells Research Articles

Fibroblast-derived exosomal microRNA regulates NKX3-1 expression in androgen-sensitive, androgen receptor-dependent prostate cancer cells.

Androgen deprivation therapy (ADT) targeting androgen production and androgen receptor (AR) signaling is the primary antihormonal therapy in the treatment of advanced prostate cancer (PCa). However, no clinically established molecular biomarkers have been identified to predict the effectiveness of ADT before starting ADT. The tumor microenvironment of PCa contains fibroblasts that regulate PCa progression by producing multiple soluble factors. We have previously reported that AR-activating factor-secreted fibroblasts increase the responsiveness of androgen-sensitive, AR-dependent PCa cells to ADT. Thus, we hypothesized that fibroblast-derived soluble factors may affect cancer cell differentiation by regulating cancer-related gene expression in PCa cells and that the biochemical characteristics of fibroblasts may be used to predict the effectiveness of ADT. Here, we investigated the effects of normal fibroblasts (PrSC cells) and three PCa patient-derived fibroblast lines (pcPrF-M5, -M28, and -M31 cells) on the expression of cancer-related genes in androgen-sensitive, AR-dependent human PCa cells (LNCaP cells) and three sublines showing different androgen sensitivities and AR dependencies. The mRNA expression of the tumor suppressor gene NKX3-1 in LNCaP cells and E9 cells (which show low androgen sensitivity and AR dependency) was significantly increased by treatment with conditioned media from PrSC and pcPrF-M5 cells but not from pcPrF-M28 and pcPrF-M31 cells. Notably, no upregulation of NKX3-1 was observed in F10 cells (AR-V7-expressing, AR-independent cells with low androgen sensitivity) and AIDL cells (androgen-insensitive, AR-independent cells). Among 81 common fibroblast-derived exosomal microRNAs that showed 0.5-fold lower expression in pcPrF-M28 and pcPrF-M31 cells than in PrSC and pcPrF-M5 cells, miR-449c-3p and miR-3121-3p were found to target NKX3-1. In only LNCaP cells, the NKX3-1 mRNA expression was significantly increased by transfection of an miR-3121-3p mimic but not that of the miR-449c-3p mimic. Thus, fibroblast-derived exosomal miR-3121-3p may be involved in preventing the oncogenic dedifferentiation of PCa cells by targeting NKX3-1 in androgen-sensitive, AR-dependent PCa cells.

Abstract 3163: Progressive prostate cancer LNCaP subclones partially retain regressive communications with patient-derived fibroblasts

Abstract The tumor microenvironment of prostate cancer (PCa) plays an important role in disease progression, responsiveness to therapy, or inversely restraint of tumor growth. PCa cell evolution including loss of androgen receptor dependency is associated with intratumor heterogeneity, generating progressive PCa cell subclones. The tumor stroma of PCa contains multiple functionally different populations of fibroblasts that regulate PCa cell progression, e.g., cancer-promoting or cancer-restraining. Fibroblasts secret a number of growth factors, cytokines, the ECM molecules, and miRNAs in a cellular characteristic-specific manner. Thus, we hypothesize that diversity of fibroblasts may induce heterogenous PCa cell progression. To investigate the heterotypical communications between PCa cells and fibroblasts, we used 3 subclones of androgen-sensitive LNCaP cells and 4 patient-derived fibroblasts. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Commercially available normal human prostate stromal cells (PrSC) was purchased from Lonza Group Ltd. Three original fibroblasts pcPrFs (-M5, -M28, -M31) were isolated from needle biopsy samples of patients with advanced PCa. To examine the effects of fibroblasts on cancer-related gene expression in PCa cells, in vitro co-culture experiment was performed using cell culture insert. In all PCa cells, LNCaP and its subclones, expressions of NFKB1, SRC, and MYC mRNA were not dramatically changed by co-culturing with 3 PCa patient-derived fibroblasts. Only in LNCaP cells, however, they were uniformly down-regulated by co-culturing with PrSC. Only in LNCaP cells, expression of CDH2 mRNA was up-regulated by co-culturing with pcPrF-M5 and pcPrF-M28. In all PCa cells, expression of NKX3-1 mRNA was uniformly up-regulated by co-culturing with pcPrF-M28 and pcPrF-M31. In LNCaP, E9, and F10 cells but not in AIDL cells, expression of GSTP1 mRNA was uniformly up-regulated by co-culturing with pcPrF-M31. Cell growth of LNCaP cells was not remarkably affected by co-culturing with pcPrF-M28 and pcPrF-M31. The heterogeneous stromal compartment in PCa tissues contains multiple functionally different populations of fibroblasts that are also associated with intratumor heterogeneity. Our data showed that up-regulations of NKX3-1 and GSTP1 mRNA expression by co-culturing with pcPrF-M28 and pcPrF-M31 were commonly observed in LNCaP subclones differing in androgen sensitivity. This result suggests that certain fibroblasts may restrain PCa cell progression by up-regulations of tumor suppressor gene expression even in progressive poorly differentiated PCa cells. Citation Format: Kenichiro Ishii, Chise Matsuda, Yasuhisa Nakagawa, Taku Shirai, Yoshifumi Hirokawa, Takeshi Sasaki, Kazuhiro Iguchi, Masatoshi Watanabe. Progressive prostate cancer LNCaP subclones partially retain regressive communications with patient-derived fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3163.

Successful Manufacturing of CAR T-Cells with Small Volume Peripheral Blood from Healthy Donors Using the Clinimacs Prodigy Device

Introduction In recent years, CAR T-cell therapy has emerged as a potentially curative intent treatment for some patients with relapsed, refractory hematologic malignancies. Despite the exciting results, not all patients are able to receive CAR T-cells due to manufacturing failures. T-cells for CAR products are typically autologous and isolated from heavily pre-treated patients, which might account for some of the manufacturing failures and suboptimal clinical efficacy. T-cells collected either early into cancer diagnosis or prior to diagnosis may improve CAR T-cell expansion and limit manufacturing failure. We evaluated the feasibility of generating a CAR T-cell product manufactured from 50 ml of healthy donor blood. Methods Collaborators at Cell Vault collected 50 ml of whole blood from 3 healthy donors, isolated peripheral blood mononuclear cells (PBMCs), and cryopreserved the cells in cryovials at 5e6/vial (1.05-1.35e8 total cells). The vials were shipped to the Medical College of Wisconsin and stored frozen in liquid nitrogen until use. All PBMC vials for a given donor were thawed and pooled. Thawed PBMCs (0.93-1.17e8 cells) were loaded onto a CliniMACS Prodigy device, CD4 and CD8 T cells enriched by immunomagnetic sorting, and T cells placed in the culture chamber with IL-7, IL-15 and TransAct reagent to induce proliferation. On the second day of manufacturing, T cells were transduced with a lentiviral CAR vector encoding anti-CD19, 4-1BB and CD3z. Final CAR T-cell products for these pre-clinical studies were harvested on day 8 of manufacture. Results Starting enriched T-cell numbers from the 3 healthy donors ranged from 4.0-4.8e7 cells, the cells were 74-79% CD4/8+, and the average CD4/CD8 ratio was 1.4. On the day of CAR T harvest (day 8), total cells in the chamber had expanded to 3.6-4.6e9 cells (74-115 fold expansion), the cells were >99% CD3+, and the average CD4/CD8 ratio was 2.9 (Table 1). Final cell numbers were similar to what previously published CAR T manufacturing runs on the CliniMACS Prodigy (Zhu et al., Cytotherapy, 2018), that started with 1x108 enriched T-cells obtained from apheresed mononuclear cells. Cell surface CD19 CAR expression on the final cell products varied from 19.2-48.1%. While more than 50% of the starting T cells had a naïve (CD62L+ CD45RO-) phenotype, the final cell products contained greater than 80% central-memory (CD62L+ CD45RO+) T cells. Finally, the number of CD19 CAR T cells obtained from these pre-clinical manufacturing runs ranged from 7.82e8 to 2.21e9 cells. Conclusions 50 ml of cryopreserved PBMCs was adequate to manufacture clinically relevant CAR T-cell therapy doses from healthy donors not previously exposed to chemotherapy. Sufficient numbers of CAR T-cells were obtained to dose an 80 kg individual with at least 9e6 cells/kg which is greater than prescribed commercial doses of CD19 CAR T-cells. Further studies are indicated to determine if T-cells collected prior to disease modifying chemotherapies result in an improved product. These results demonstrate feasibility for generating CAR T cells from small volumes of whole blood collected at a time point before a cancer patient has been treated with multiple lines of therapy that could negatively impact starting T cell numbers and function. Disclosures Hari: GSK: Consultancy; Amgen: Consultancy; BMS: Consultancy; Takeda: Consultancy; Incyte Corporation: Consultancy; Janssen: Consultancy. Shah:TG Therapeutics: Consultancy; Celgene: Consultancy, Honoraria; Incyte: Consultancy; Kite Pharma: Consultancy, Honoraria; Cell Vault: Research Funding; Miltenyi Biotec: Honoraria, Research Funding; Lily: Consultancy, Honoraria; Verastim: Consultancy. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding.

Loss of Fibroblast-Dependent Androgen Receptor Activation in Prostate Cancer Cells is Involved in the Mechanism of Acquired Resistance to Castration.

Loss of androgen receptor (AR) dependency in prostate cancer (PCa) cells is associated with progression to castration-resistant prostate cancer (CRPC). The tumor stroma is enriched in fibroblasts that secrete AR-activating factors. To investigate the roles of fibroblasts in AR activation under androgen deprivation, we used three sublines of androgen-sensitive LNCaP cells (E9 and F10 cells: low androgen sensitivity; and AIDL cells: androgen insensitivity) and original fibroblasts derived from patients with PCa. We performed in vivo experiments using three sublines of LNCaP cells and original fibroblasts to form homotypic tumors. The volume of tumors derived from E9 cells plus fibroblasts was reduced following androgen deprivation therapy (ADT), whereas that of F10 or AIDL cells plus fibroblasts was increased even after ADT. In tumors derived from E9 cells plus fibroblasts, serum prostate-specific antigen (PSA) decreased rapidly after ADT, but was still detectable. In contrast, serum PSA was increased even in F10 cells inoculated alone. In indirect cocultures with fibroblasts, PSA production was increased in E9 cells. Epidermal growth factor treatment stimulated Akt and p44/42 mitogen-activated protein kinase phosphorylation in E9 cells. Notably, AR splice variant 7 was detected in F10 cells. Overall, we found that fibroblast-secreted AR-activating factors modulated AR signaling in E9 cells after ADT and loss of fibroblast-dependent AR activation in F10 cells may be responsible for CRPC progression.

Abstract 2026: Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer

Abstract Well-differentiated prostate cancer (PCa) cells are generally androgen/androgen receptor (AR)-dependent, i.e. AR signaling regulates cell cycle and differentiation. Loss of AR signaling after androgen deprivation therapy (ADT) triggers AR-independent outgrowth, generating poorly differentiated uncontrollable PCa cells. To prevent development and progression of castration-resistant prostate cancer (CRPC), we hypothesize that preservation of AR signaling after ADT is an important target. Tumor stroma surrounding PCa cells is enriched in fibroblasts secreting AR-activating factors, e.g. EGF, IGF1, and IL-6. Thus, we investigated the role of fibroblasts in AR activation of human prostate cancer LNCaP sublines differing in androgen sensitivity under androgen deprivation. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Original fibroblasts pcPrFs (-M5, -M6, -M7) were isolated from needle biopsy samples of patients with PCa. The PCa cells alone or PCa cells plus pcPrF-M5 were grafted beneath the renal capsule of male athymic nude mice, and then mice were castrated on 14 days post transplantation. Tumor volume of E9 cells + pcPrF-M5 became diminished post castration but that of F10 and AIDL cells + pcPrF-M5 was gradually increased even post castration. In E9 cells + pcPrF-M5 tumors, serum PSA became detected on day 21 post castration. Interestingly, serum PSA was gradually increased even in F10 cells inoculated alone. In AIDL tumors with or without pcPrF-M5, serum PSA was not detected because of mutated AR in AIDL cells. Cell growth of E9 and AIDL cells was directly increased by treating with EGF, whereas that in F10 cells was not affected. Phosphorylation of AKT and ERK1/2 in E9 and AIDL cells was also significantly increased by treating with EGF, whereas that in F10 cells was not affected. In the condition of co-cultures with fibroblasts, PSA production was directly increased in E9 cells but not in F10 and AIDL cells. Protein expression of AR splice variant 7 (AR-V7) was only detected in F10 cells. The reduced AR-dependency of PCa cells is an important clinical development because of its association with the cell’ progression to CRPC. Here our results showed that fibroblasts secreting AR-activating factors preserved AR signaling in E9 cells after ADT, indicating that this type of PCa cells can be controlled by ADT. In contrast, we suggest that loss of fibroblasts-dependent AR activation in F10 and AIDL cells may be responsible for development and progression of CRPC. Citation Format: Kenichiro Ishii, Izumi Matsuoka, Takeshi Sasaki, Manabu Kato, Kohei Nishikawa, Hideki Kanda, Yoshifumi Hirokawa, Kazuhiro Iguchi, Kiminobu Arima, Masatoshi Watanabe, Yoshiki Sugimura. Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2026.

Pirfenidone, an Anti-Fibrotic Drug, Suppresses the Growth of Human Prostate Cancer Cells by Inducing G1 Cell Cycle Arrest

Pirfenidone (PFD) is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis by inducing G1 cell cycle arrest in fibroblasts. We hypothesize that PFD can induce G1 cell cycle arrest in different types of cells, including cancer cells. To investigate the effects of PFD treatment on the growth of human prostate cancer (PCa) cells, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines (androgen-low-sensitive E9 and F10 cells and androgen-insensitive AIDL cells), as well as an androgen-insensitive human PCa cell line (PC-3). PFD treatment suppressed the growth of all PCa cells. Transforming growth factor β1 secretion was significantly increased in PFD-treated PCa cells. In both LNCaP and PC-3 cells, PFD treatment increased the population of cells in the G0/G1 phase, which was accompanied by a decrease in the S/G2 cell population. CDK2 protein expression was clearly decreased in PFD-treated LNCaP and PC-3 cells, whereas p21 protein expression was increased in only PFD-treated LNCaP cells. In conclusion, PFD may serve as a novel therapeutic drug that induces G1 cell cycle arrest in human PCa cells independently of androgen sensitivity. Thus, in the tumor microenvironment, PFD might target not only fibroblasts, but also heterogeneous PCa cells of varying androgen-sensitivity levels.

Interleukin-6 induces VEGF secretion from prostate cancer cells in a manner independent of androgen receptor activation.

The reduced androgen-sensitivity of prostate cancer (PCa) cells is an important clinical development because of its association with the cells' progression to castration-resistant prostate cancer (CRPC). During androgen deprivation therapy (ADT), stroma-derived growth factors and cytokines can activate the androgen receptor (AR). For example, IL-6 is a multifunctional cytokine that is involved in the malignancy of PCa cells through AR activation. In the present study, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines to investigate the relationship between the responsiveness of PCa cells to IL-6 treatment and the cellular AR signaling pathway. The androgen-low-sensitive F10 and E9 cells were obtained from LNCaP cells by limiting dilution method in regular culture condition. In contrast, the androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging in hormone-depleted condition. Original carcinoma-associated fibroblasts (CAFs) PCaSC-8 and PCaSC-9 cells were isolated from needle biopsy samples of PCa patients. In fibroblasts derived from PCa patients, IL-6 secretion was generally higher than that observed with normal fibroblasts. In contrast, IL-6 secretion was not detected in LNCaP and its sublines. The soluble IL-6 receptor was detected in PCa cells but not in fibroblasts. IL-6 treatment suppressed cell growth of LNCaP, F10, and E9 cells but not AIDL cells and it was accompanied with neuroendocrine-like differentiation. Induction of PSA secretion was observed in IL-6-treated LNCaP and F10 cells. VEGF secretion was strongly induced in IL-6-treated LNCaP and AIDL cells. IL-6-induced VEGF secretion was significantly suppressed by a PI3K inhibitor (LY294002) and it was accompanied by inhibited phosphorylation of Akt. Our results suggest that IL-6 might induce VEGF secretion from PCa cells in a manner independent of AR activation. To prevent IL-6-induced VEGF secretion, inhibition of the PI3K/AKT signaling pathway could be an important pharmacological goal regardless of ADT.

Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia

GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded >1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

788 ABERRANT ACTIVATION OF PARACRINE LOOP BETWEEN PROSTATE CANCER CELLS AND CARCINOMA-ASSOCIATED FIBROBLASTS BY OVEREXPRESSION OF STROMAL INTERLEUKIN-6

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2012788 ABERRANT ACTIVATION OF PARACRINE LOOP BETWEEN PROSTATE CANCER CELLS AND CARCINOMA-ASSOCIATED FIBROBLASTS BY OVEREXPRESSION OF STROMAL INTERLEUKIN-6 Kenichiro Ishii, Yoichi Iwamoto, Manabu Kato, Kazuhiro Iguchi, Atsushi Mizokami, Kiminobu Arima, Taizo Shiraishi, and Yoshiki Sugimura Kenichiro IshiiKenichiro Ishii Tsu, Japan More articles by this author , Yoichi IwamotoYoichi Iwamoto Tsu, Japan More articles by this author , Manabu KatoManabu Kato Tsu, Japan More articles by this author , Kazuhiro IguchiKazuhiro Iguchi Gifu, Japan More articles by this author , Atsushi MizokamiAtsushi Mizokami Ishikawa, Japan More articles by this author , Kiminobu ArimaKiminobu Arima Tsu, Japan More articles by this author , Taizo ShiraishiTaizo Shiraishi Tsu, Japan More articles by this author , and Yoshiki SugimuraYoshiki Sugimura Tsu, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.876AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In the tumor microenvironment, activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. During primary cancer progression, cancer cells generate the carcinoma-associated fibroblasts (CAFs) in adjacent normal fibroblasts, and then CAFs support the proliferation and invasion of adjacent cancer cells in a paracrine fashion. The serum level of interleukin-6 (IL-6) is a significant prognostic factor for prostate cancer (PCa) patients with disseminated diseases, suggesting positive roles of IL-6 in the aberrant activation of paracrine loop between PCa cells and CAFs. In this study, we investigated the relationship between responsiveness of PCa cells to stromal IL-6 and cellular AR signaling. METHODS The androgen-low-sensitive E9 cells were obtained from androgen-sensitive LNCaP cell population by limiting dilution method in regular culture condition. In contrast, the androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging in hormone-depleted condition. PCaSC-8 and PCaSC-9 cells were primary cultured from different human PCa specimens. RESULTS IL-6 and VEGF protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than normal human prostate stromal cells PrSC. In contrast to stromal cells, no IL-6 secretion from LNCaP, E9, and AIDL was detected. IL-6 treatment (10 ng/mL) suppressed cell proliferation of LNCaP and E9 accompanied with changes of cell morphology, i.e. induction of neuroendocrine-like differentiation. Induction of PSA secretion was observed only in IL-6-treated LNCaP. VEGF secretion was significantly increased in both IL-6-treated LNCaP and AIDL. A selective alpha1-adrenoceptor antagonist naftopidil (10 μM) but not tamsulosin suppressed IL-6-induced VEGF secretion in AIDL. CONCLUSIONS In this study, we have shown the evidence that secretions of IL-6 and VEGF from primary cultured CAFs derived PCa patients are generally higher than normal fibroblasts. Our results suggest that overexpression of stromal IL-6 might increase VEGF secretion from adjacent PCa cells in a paracrine fashion. Using LNCaP sublines, E9 and AIDL, differing in androgen-sensitivity, we demonstrated here that IL-6-induced VEGF secretion from PCa cells might be through cellular AR signaling-independent pathway. Identifying mechanisms underlying the aberrant activation of paracrine loop between PCa cells and CAFs is an initial step toward designing CAF-targeted therapy for treatment of PCa. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e322-e323 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kenichiro Ishii Tsu, Japan More articles by this author Yoichi Iwamoto Tsu, Japan More articles by this author Manabu Kato Tsu, Japan More articles by this author Kazuhiro Iguchi Gifu, Japan More articles by this author Atsushi Mizokami Ishikawa, Japan More articles by this author Kiminobu Arima Tsu, Japan More articles by this author Taizo Shiraishi Tsu, Japan More articles by this author Yoshiki Sugimura Tsu, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Heterogenous induction of carcinoma-associated fibroblast-like differentiation in normal human prostatic fibroblasts by co-culturing with prostate cancer cells

In the tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are considered to play a critical role in the promotion of tumorigenesis. However, the mechanisms that generate CAFs are not well elucidated. To understand how CAFs are generated during primary cancer progression, we investigated the biochemical characteristics of normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer (PCa) cells in vitro. In primary cultures of human PCa-derived stromal cells (PCaSC-8 and PCaSC-9), expression of TNC, ACTA2, EGF, FGF7, and IGF1 mRNA was generally higher than PrSC but gene expression patterns were not uniform between PCaSC-8 and PCaSC-9 cells. Transforming growth factor β (TGFβ) and vascular endothelial growth factor (VEGF) protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than PrSC but levels of both secreted proteins were not same. When PrSCs were co-cultured with androgen-sensitive LNCaP cells or its sublines, androgen-low-sensitive E9 cells and androgen-insensitive AIDL cells, mRNA expression of IGF1 was significantly increased in all combinations. In contrast, expression of COL1A1, TNC, and ACTA2 mRNA was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Protein production of VEGF was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Increase of TGFβ protein was observed only in E9 + PrSC co-cultures. These biochemical characteristics of PrSC were partially recapitulated in TGFβ-treated PrSC. We have demonstrated that normal fibroblasts co-cultured with cancer cells become activated and exhibit biochemical characteristics of CAFs in a heterogenous manner. Our results suggest that heterogenous induction of CAF-like differentiation might be strongly dependent on biochemical characteristics of adjacent cancer cells.

Naftopidil, a Selective α1-Adrenoceptor Antagonist, Suppresses Human Prostate Tumor Growth by Altering Interactions between Tumor Cells and Stroma

In prostate cancer, tumor-stroma interactions play a critical role in the promotion of tumorigenesis, and thus the prevention of those interactions is a promising target to suppress tumor growth. Several studies demonstrated that alpha(1)-adrenoceptor (α(1)-AR) antagonists, therapeutic drugs for benign prostatic hyperplasia, have growth inhibitory effects on human prostate cancer (PCa) cells through induction of apoptosis or G(1) cell-cycle arrest. However, their direct actions on stromal cells surrounding cancer cells have not yet been elucidated. In this study, we investigated the effects of subtype-selective α(1)-AR antagonists (naftopidil, tamsulosin, and silodosin) on prostate tumor growth with a focus on the role of stroma, using commercially available fibroblast cells (PrSC). Tumorigenic studies in vivo showed significant reductions in tumor growth when E9 cells (an androgen low-sensitive LNCaP subline) grafted with PrSC were treated with naftopidil. In in vitro analyses, naftopidil and silodosin showed antiproliferative effects on PCa cells regardless of androgen sensitivity and α(1)-AR subtype expression. In PrSC, a strong growth inhibitory effect was observed with naftopidil but not silodosin. Flow cytometric analysis revealed that naftopidil, but not silodosin, induced G(1) cell-cycle arrest in both PCa cells and PrSC. In naftopidil-treated PrSC, total interleukin-6 protein was significantly reduced with increased suppression of cell proliferation. Silodosin induced weak early apoptosis only in PCa cells. These findings demonstrated that naftopidil strongly suppressed cell proliferation of stromal cells, resulting in decreased tumorigenic soluble factor, suggesting that naftopidil might be effective in preventing stromal support of tumor cells.

690 UNIQUE GROWTH-INHIBITORY EFFECTS OF SUBTYPE SELECTIVE α1-ADRENOCEPTOR ANTAGONISTS ON HUMAN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2010690 UNIQUE GROWTH-INHIBITORY EFFECTS OF SUBTYPE SELECTIVE α1-ADRENOCEPTOR ANTAGONISTS ON HUMAN PROSTATE CANCER CELLS Yasuhide Hori, Kenichiro Ishii, Hideki Kanda, Kouhie Nishikawa, Yuko Yoshio, Norihito Soga, Hideaki Kise, Kiminobu Arima, and Yoshiki Sugimura Yasuhide HoriYasuhide Hori More articles by this author , Kenichiro IshiiKenichiro Ishii More articles by this author , Hideki KandaHideki Kanda More articles by this author , Kouhie NishikawaKouhie Nishikawa More articles by this author , Yuko YoshioYuko Yoshio More articles by this author , Norihito SogaNorihito Soga More articles by this author , Hideaki KiseHideaki Kise More articles by this author , Kiminobu ArimaKiminobu Arima More articles by this author , and Yoshiki SugimuraYoshiki Sugimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1089AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It has been well established that α1-adrenoceptor (α1-AR) antagonists, therapeutic drugs for BPH, have growth-inhibitory effects on human prostate cancer (hPCa) cells. In Japan, subtype selective α1-AR antagonists are frequently used because of low incidence of side effects. Although we have recently reported the growth-inhibitory effects of the α1d-selective antagonist naftopidil on hPCa cells, little is known about the relationship between the growth-inhibitory effects of naftopidil and the androgen receptor signal or the α1-AR signal in hPCa cells. In this study, we investigated the biochemical mechanisms of growth-inhibitory effects by subtype selective α1-AR antagonists in terms of androgen sensitivity of hPCa cells and α1-AR selectivity of each antagonist. METHODS The three α1a selective-AR antagonists (tamsulosin, silodosin, and RS100329) and two α1d selective antagonists (naftopidil and BMY7378) were used. We first evaluated the effects of each antagonist (10 μM) on the cell proliferation of LNCaP and the derived two sublines E9 and AIDL differing in hormone sensitive status. Next, to simulate the tumor microenvironment in an in vivo transplant model, we recombined E9 and human prostatic stromal cells (PrSC) in collagen gels and grafted them beneath the renal capsule of athymic mice. Mice were treated with each antagonist (10 mg/Kg/day) by oral gavage. RESULTS The parental LNCaP, androgen-low-sensitive E9, and androgen-insensitive AIDL expressed mRNAs of both α1a- and α1d-subtype, mainly α1d-subtype, and mainly α1a-subtype, respectively. Silodosin and naftopidil but not tamsulosin showed approximately equivalent growth-inhibitory effects on LNCaP, E9, and AIDL cells in vitro. FACS analysis revealed induction of apoptosis in silodosin-treated hPCa cells and increase of G1 arrest in naftopidil-treated hPCa cells. Tumorigenic study in vivo showed that significant reduction of E9 + PrSC tumor weight was observed in mice treated with not only silodosin and naftopidil but also tamsulosin. CONCLUSIONS We have demonstrated that subtype selective α1-AR antagonists have unique growth-inhibitory effects on hPCa cells. The growth-inhibitory effects of subtype selective α1-AR antagonists are not related to both androgen sensitivity of hPCa cells and α1-AR subtype expression in each hPCa cells, suggesting that subtype selective α1-AR antagonists are potentially effective to prevent not only development of hPCa but also recurrence of hPCa. Tsu City, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e269-e270 Peer Review Report Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yasuhide Hori More articles by this author Kenichiro Ishii More articles by this author Hideki Kanda More articles by this author Kouhie Nishikawa More articles by this author Yuko Yoshio More articles by this author Norihito Soga More articles by this author Hideaki Kise More articles by this author Kiminobu Arima More articles by this author Yoshiki Sugimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Evidence that androgen-independent stromal growth factor signals promote androgen-insensitive prostate cancer cell growth in vivo

Activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. To discover new therapeutic targets for hormone-refractory prostate tumor growth under androgen ablation therapy, androgen-sensitive LNCaP cells and the derived sublines, E9 (androgen-low-sensitive), and AIDL (androgen-insensitive), were recombined with androgen-dependent embryonic rat urogenital sinus mesenchyme (UGM). Tumors of E9 + UGM and AIDL + UGM were approximately three times as large as those of LNCaP + UGM. Tumors grown in castrated hosts exhibited reduced growth as compared with those in intact hosts. However, in castrated hosts, E9 + UGM and AIDL + UGM tumors were still approximately twice as large as those of LNCaP + UGM. Cell proliferation in tumors of E9 + UGM and AIDL + UGM grown in castrated host, was significantly higher than that in tumors of LNCaP + UGM. In vitro, expression of fibroblast growth factor (FGF)-2 and IGF-I, but not FGF-7 mRNA, was significantly reduced in UGM under androgen starvation. In cell culture, E9 cells were responsive to FGF-2 and FGF-7 stimulation, while AIDL responded to FGF-7 and IGF-1. Expression of FGFR1 and FGFR2 was considerably higher in E9 than those in LNCaP, similarly expression of FGFR2 and IGF-IR were elevated in AIDL. These data suggest that activation of prostate cancer cell growth through growth factor receptor expression may result in the activity of otherwise androgen-independent stromal growth factor signals such as FGF-7 under conditions of androgen ablation.

Isolation and Characterization of LNCaP Sublines Differing in Hormone Sensitivity

Prostate cancer is a heterogeneous disease with varying degrees of androgen sensitivity. In this study, we performed a limiting dilution of human prostate LNCaP cells, and isolated two sublines, LNCaP-E9 and LNCaP-G4, with differential hormone-sensitivity. Two LNCaP sublines were obtained by the limiting dilution method. The growth of E9 cells was decreased in the presence of androgens, while that of androgen-treated G4 cells was biphasic. Although the androgen receptor expression level in E9 cells was similar to that seen in G4 cells, the expression of PSA mRNA and protein was significantly lower in the E9 cells. Moreover, the androgen-based stimulation of PSA mRNA expression was less sensitive in E9 cells than G4 cells. Intracellular zinc level did not differ between E9 and G4 cells, but ZnT3 mRNA expression was significantly higher in the E9 cells. When the cells were grafted at the subrenal capsule, the number of CD31-positive vessels with a lumen was approximately 2.5 times higher than that in G4 tumors. LNCaP-E9 cells show lower androgen sensitivity than LNCaP-G4 cells. E9 and G4 cells would be helpful for understanding the biology of hormone-refractory prostate cancer.

Immunochemical and Proteomic Analysis of Covalent Adducts Formed by Quinone Methide Tumor Promoters in Mouse Lung Epithelial Cell Lines

Two quinone methide (QM) metabolites of the phenolic antioxidant butylated hydroxytoluene (BHT), 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and the tert-butyl-hydroxylated derivative (BHTOH-QM), are believed to be responsible for promoting lung tumor formation in mice treated with BHT. QMs are strongly electrophilic and undergo Michael type additions with nucleophiles at the exocyclic methylene to form benzylic thioether adducts. Our goal was to identify intracellular protein targets of these QMs in order to gain insight into their effects on tumorigenesis. Cell line E10 of mouse lung epithelial origin and its spontaneous transformant, the tumorigenic E9 cell line, were treated with BHT-QM or BHTOH-QM, and cellular proteins were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Adducted proteins were detected on western blots with polyclonal antibodies developed to a conjugate of BHTOH-QM that recognized adducts of both QMs bound to thiol groups of Cys and side chain amino groups of Lys and His residues. Tryptic digests of immunoreactive proteins were analyzed by HPLC mass spectrometry (LC/MS) and identified by searching protein databases using MS/MS data. In a few cases, adducted peptides in these digests were detected by matrix-assisted laser desorption/ionization time-of-flight MS. A total of 37 immunoreactive proteins were identified including proteins involved in carbohydrate metabolism, nucleic acid synthesis, and RNA and protein processing, in addition to several cytoskeletal and stress-related proteins. About half of the protein adducts were found in both cell lines. Adducts detected only in transformed E9 cells include glutathione S-transferase P1, peroxiredoxin 2, nucleoside diphosphate kinase, and vinculin, whereas several alkylated cytoskeletal proteins such as tubulins, vimentin, calvasculin, and calcyclin were detected exclusively in E10 cells. Several of the proteins modified by BHT-derived QMs have been implicated in various aspects of tumorigenesis and are excellent candidates for further study into the consequences of alkylation on cellular transformation.

Ethanol upregulates pro-fibrogenic connective tissue growth factor (CTGF) gene expression in HepG2 cells via cytochrome P450 2E1-mediated ethanol oxidation

Connective tissue growth factor (CTGF) is a pro-fibrogenic molecule involved in several human fibrotic disorders. CTGF overexpression in the liver, through upregulation of CTGF mRNA in hepatic stellate cells (HSCs), is reported to correlate with the degree of fibrosis. Although alcohol is a major cause of hepatic fibrosis, the role of CTGF in alcoholic hepatic fibrogenesis, has been poorly understood. Oxidative stress, mediated by ethanol-inducible Cytochrome P-4502E1(CYP2E1), has been implicated as a crucial factor in alcoholic hepatic fibrogenesis. Therefore, we investigated the contribution of CYP2E1-mediated ethanol oxidation to CTGF mRNA expression by using a well-established HepG2 cell line constitutively expressing CYP2E1 (E9 cells). CTGF mRNA quantitation by a real-time reverse polymerase chain reaction (RT-PCR) showed a significant increase in CTGF mRNA levels in ethanol-treated E9 cells. Phorbol myristate acetate (PMA), which enhances CYP2E1 expression, increased CTGF mRNA levels. This increase was diminished after co-incubation with 4-methylpyrazole (4MP), a CYP2E1 inhibitor, or an antioxidant, N -acetylcysteine. We visualized the state of oxidative stress only in ethanol-treated E9 cells by a newly-established anti-acrolein-modified antibody. In conclusion, our study has first identified a possible role of CYP2E1-mediated ethanol oxidation in CTGF gene upregulation, suggesting a considerable role of CTGF in alcoholic hepatic fiberogenesis.

Regulation of lung epithelial cell morphology by cAMP-dependent protein kinase type I isozyme.

Cell shape is mediated in part by the actin cytoskeleton and the actin-binding protein vinculin. These proteins in turn are regulated by protein phosphorylation. We assessed the contribution of cAMP-dependent protein kinase A isozyme I (PKA I) to lung epithelial morphology using the E10/E9 sibling cell lines. PKA I concentration is high in flattened, nontumorigenic E10 cells but low in their round E9 transformants. PKA I activity was lowered in E10 cells by stable transfection with a dominant negative RIalpha mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Calpha catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and grew in colonies, their actin microfilaments were disrupted, and vinculin localization at cell-cell junctions was diminished. The converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in lung cancer would provide useful adjuvant therapy.

Growth inhibition in G(1) and altered expression of cyclin D1 and p27(kip-1 )after forced connexin expression in lung and liver carcinoma cells.

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G(1)-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G(1)-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27(kip-1) protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-beta, epidermal growth factor receptor, protein kinase C-alpha, protein kinase A regulatory subunit-Ialpha, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G(1) growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27(kip-1), but not with changes in other growth-related functions.

Cytokine-induced nitric oxide formation in normal but not in neoplastic murine lung epithelial cell lines.

Cytomix, a mixture of interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta, induces nitric oxide (NO) production in lung epithelial cell lines. It is not known whether neoplastic transformation alters a cell's ability to form NO in response to cytokines. The present study investigated NO formation in two murine lines of immortalized "normal" (nontumorigenic) lung epithelial cells of alveolar type II origin, E10 and C10, and their sibling spontaneous transformants, E9 and A5. Nontumorigenic cells elaborated much more NO after cytomix exposure than did their tumorigenic counterparts. NO production was prevented by inhibiting protein synthesis and NO synthase and attenuated by dexamethasone. Northern and Western blot analyses of inducible NO synthase (iNOS) demonstrated cytomix-induced induction of iNOS only in nontumorigenic cells. The deficiency in NO production in tumorigenic cells was not associated with reduced iNOS mRNA stability or with differences in cytomix-induced nuclear factor-kappaB activation. Although cytomix caused a greater production of NO in E10 cells than in E9 cells, the same treatment induced equivalent proliferation in both cell lines. These results indicate a specific deficiency in cytokine-induced NO synthesis in transformed murine lung epithelial cells relative to their normal progenitor cells and provide a model for investigating iNOS regulation.

Role of Gap Junctions in Lung Neoplasia

Reduced gap junctional intercellular communication (GJIC) has been noted in many types of neoplastic cells and may contribute to the neoplastic phenotype. This study assessed GJIC (by fluorescent dye-coupling) and gap junction protein (connexin) expression in mouse and human lung carcinoma cell lines and investigated whether reduced GJIC was involved in their neoplastic phenotype. Dye-coupling and connexin43 (Cx43) expression were much lower in most of the carcinoma lines (16 of 22) compared to nontransformed lung epithelial cells. Other connexins were not detected. A poorly communicating mouse lung carcinoma cell line (E9) was transfected with Cx43 or transduced with Cx32 and several stable clones were isolated that had 2- to 4-fold increased dye coupling. When evaluated for growth in vitro, the population doubling times were increased and the saturation densities were decreased in the clones. When assessed for tumorigenicity, the parental E9 cells formed tumors with a 100% incidence (6/6 mice), whereas the clones varied in tumorigenic response (0-88% incidence). The best communicating clone (E9-2) was not tumorigenic. The highly communicating Cx32 clone, E9/32-9, gave a tumor incidence of 88%. These results suggest that restoration of GJIC by forced connexin expression can reduce the growth and tumorigenicity of lung carcinoma cells in a connexin-specific manner.