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Background: Single drug use has not achieved satisfactory results in the treatment of prostate cancer, despite application of increasingly widespread targeted therapeutics. In the present study, the combined impact of the mammalian target of rapamycin (mTOR)-inhibitor RAD001, the dual EGFr and VGEFr tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer growth and adhesion in vitro was investigated. Methods: PC-3, DU-145 and LNCaP cells were treated with RAD001, AEE788 or VPA or with a RAD-AEE-VPA combination. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT-assay, flow cytometry and western blotting, respectively. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins as well as migratory properties of the cells was evaluated, and integrin alpha and beta subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined. Results: All drugs, separately applied, reduced tumor cell adhesion, migration and growth. A much stronger anti-cancer effect was evoked by the triple drug combination. Particularly, cdk1, 2 and 4 and cyclin B were reduced, whereas p27 was elevated. In addition, simultaneous application of RAD001, AEE788 and VPA altered the membranous, cytoplasmic and gene expression pattern of various integrin alpha and beta subtypes, reduced integrin-linked kinase (ILK) and deactivated focal adhesion kinase (FAK). Signaling analysis revealed that EGFr and the downstream target Akt, as well as p70S6k was distinctly modified in the presence of the drug combination. Conclusions: Simultaneous targeting of several key proteins in prostate cancer cells provides an advantage over targeting a single pathway. Since strong anti-tumor properties became evident with respect to cell growth and adhesion dynamics, the triple drug combination might provide progress in the treatment of advanced prostate cancer.
Background: Although mechanistic target of rapamycin (mTOR) inhibitors, such as temsirolimus, show promise in treating bladder cancer, acquired resistance often hampers efficacy. This study evaluates mechanisms leading to resistance. Methods: Cell growth, proliferation, cell cycle phases, and cell cycle regulating proteins were compared in temsirolimus resistant (res) and sensitive (parental—par) RT112 and UMUC3 bladder cancer cells. To evaluate invasive behavior, adhesion to vascular endothelium or to immobilized extracellular matrix proteins and chemotactic activity were examined. Integrin α and β subtypes were analyzed and blocking was done to evaluate physiologic integrin relevance. Results: Growth of RT112res could no longer be restrained by temsirolimus and was even enhanced in UMUC3res, accompanied by accumulation in the S- and G2/M-phase. Proteins of the cdk-cyclin and Akt-mTOR axis increased, whereas p19, p27, p53, and p73 decreased in resistant cells treated with low-dosed temsirolimus. Chemotactic activity of RT112res/UMUC3res was elevated following temsirolimus re-exposure, along with significant integrin α2, α3, and β1 alterations. Blocking revealed a functional switch of the integrins, driving the resistant cells from being adhesive to being highly motile. Conclusion: Temsirolimus resistance is associated with reactivation of bladder cancer growth and invasive behavior. The α2, α3, and β1 integrins could be attractive treatment targets to hinder temsirolimus resistance.
Insulin-like growth factor-1 (IGF-1)-related signaling is associated with prostate cancer progression. Links were explored between IGF-1 and expression of integrin adhesion receptors to evaluate relevance for growth and migration. Androgen-resistant PC3 and DU145 and androgen-sensitive LNCaP and VCaP prostate cancer cells were stimulated with IGF-1 and tumor growth (all cell lines), adhesion and chemotaxis (PC3, DU145) were determined. Evaluation of Akt/mTOR-related proteins, focal adhesion kinase (FAK) and integrin α and β subtype expression followed. Akt knock-down was used to investigate its influence on integrin expression, while FAK blockade served to evaluate its influence on mTOR signaling. Integrin knock-down served to investigate its influence on tumor growth and chemotaxis. Stimulation with IGF-1 activated growth in PC3, DU145, and VCaP cells, and altered adhesion and chemotactic properties of DU145 and PC3 cells. This was associated with time-dependent alterations of the integrins α3, α5, αV, and β1, FAK phosphorylation and Akt/mTOR signaling. Integrin blockade or integrin knock-down in DU145 and PC3 cells altered tumor growth, adhesion, and chemotaxis. Akt knock-down (DU145 cells) cancelled the effect of IGF-1 on α3, α5, and αV integrins, whereas FAK blockade cancelled the effect of IGF-1 on mTOR signaling (DU145 cells). Prostate cancer growth and invasion are thus controlled by a fine-tuned network between IGF-1 driven integrin-FAK signaling and the Akt-mTOR pathway. Concerted targeting of integrin subtypes along with Akt-mTOR signaling could, therefore, open options to prevent progressive dissemination of prostate cancer.
The mechanistic target of rapamycin (mTOR) is elevated in prostate cancer, making this protein attractive for tumor treatment. Unfortunately, resistance towards mTOR inhibitors develops and the tumor becomes reactivated. We determined whether epigenetic modulation by the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), may counteract non-responsiveness to the mTOR inhibitor, temsirolimus, in prostate cancer (PCa) cells. Prostate cancer cells, sensitive (parental) and resistant to temsirolimus, were exposed to VPA, and tumor cell growth behavior compared. Temsirolimus resistance enhanced the number of tumor cells in the G2/M-phase, correlating with elevated cell proliferation and clonal growth. The cell cycling proteins cdk1 and cyclin B, along with Akt-mTOR signaling increased, whereas p19, p21 and p27 decreased, compared to the parental cells. VPA significantly reduced cell growth and up-regulated the acetylated histones H3 and H4. Cdk1 and cyclin B decreased, as did phosphorylated mTOR and the mTOR sub-complex Raptor. The mTOR sub-member Rictor and phosphorylated Akt increased under VPA. Knockdown of cdk1, cyclin B, or Raptor led to significant cell growth reduction. HDAC inhibition through VPA counteracts temsirolimus resistance, probably by down-regulating cdk1, cyclin B and Raptor. Enhanced Rictor and Akt, however, may represent an undesired feedback loop, which should be considered when designing future therapeutic regimens.
Background: Targeted therapies have improved therapeutic options of treating renal cell carcinoma (RCC). However, drug response is temporary due to resistance development.
Methods: Functional and molecular changes in RCC Caki-1 cells, after acquired resistance to the mammalian target of rapamycin (mTOR)-inhibitor everolimus (Cakires), were investigated with and without additional application of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA). Cell growth was evaluated by MTT assay, cell cycle progression and apoptosis by flow cytometry. Target molecules of everolimus and VPA, apoptotic and cell cycle regulating proteins were investigated by western blotting. siRNA blockade was performed to evaluate the functional relevance of the proteins.
Results: Everolimus resistance was accompanied by significant increases in the percentage of G2/M-phase cells and in the IC50. Akt and p70S6K, targets of everolimus, were activated in Cakires compared to drug sensitive cells. The most prominent change in Cakires cells was an increase in the cell cycle activating proteins cdk2 and cyclin A. Knock-down of cdk2 and cyclin A caused significant growth inhibition in the Cakires cells. The HDAC-inhibitor, VPA, counteracted everolimus resistance in Cakires, evidenced by a significant decrease in tumor growth and cdk2/cyclin A.
Conclusion: It is concluded that non-response to everolimus is characterized by increased cdk2/cyclin A, driving RCC cells into the G2/M-phase. VPA hinders everolimus non-response by diminishing cdk2/cyclin A. Therefore, treatment with HDAC-inhibitors might be an option for patients with advanced renal cell carcinoma and acquired everolimus resistance.
This study was designed to investigate whether epigenetic modulation by histone deacetylase (HDAC) inhibition might circumvent resistance towards the mechanistic target of rapamycin (mTOR) inhibitor temsirolimus in a prostate cancer cell model. Parental (par) and temsirolimus-resistant (res) PC3 prostate cancer cells were exposed to the HDAC inhibitor valproic acid (VPA), and tumor cell adhesion, chemotaxis, migration, and invasion were evaluated. Temsirolimus resistance was characterized by reduced binding of PC3res cells to endothelium, immobilized collagen, and fibronectin, but increased adhesion to laminin, as compared to the parental cells. Chemotaxis, migration, and invasion of PC3res cells were enhanced following temsirolimus re-treatment. Integrin α and β receptors were significantly altered in PC3res compared to PC3par cells. VPA significantly counteracted temsirolimus resistance by down-regulating tumor cell–matrix interaction, chemotaxis, and migration. Evaluation of integrin expression in the presence of VPA revealed a significant down-regulation of integrin α5 in PC3res cells. Blocking studies demonstrated a close association between α5 expression on PC3res and chemotaxis. In this in vitro model, temsirolimus resistance drove prostate cancer cells to become highly motile, while HDAC inhibition reversed the metastatic activity. The VPA-induced inhibition of metastatic activity was accompanied by a lowered integrin α5 surface level on the tumor cells.
Focal therapy is a modern alternative to selectively treat a specific part of the prostate harboring clinically significant disease while preserving the rest of the gland. The aim of this therapeutic approach is to retain the oncological benefit of active treatment and to minimize the side-effects of common radical treatments. The oncological effectiveness of focal therapy is yet to be proven in long-term robust trials. In contrast, the toxicity profile is well-established in randomized controlled trials and multiple robust prospective cohort studies. This narrative review summarizes the relevant evidence on complications and their management after focal therapy. When compared to whole gland treatments, focal therapy provides a substantial benefit in terms of adverse events reduction and preservation of genito-urinary function. The most common complications occur in the peri-operative period. Urinary tract infection and acute urinary retention can occur in up to 17% of patients, while dysuria and haematuria are more common. Urinary incontinence following focal therapy is very rare (0–5%), and the vast majority of patients recover in few weeks. Erectile dysfunction can occur after focal therapy in 0–46%: the baseline function and the ablation template are the most important factors predicting post-operative erectile dysfunction. Focal therapy in the salvage setting after external beam radiotherapy has a significantly higher rate of complications. Up to one man in 10 will present a severe complication.
Background: Regulatory T cells (Treg) expressing the transcription factor forkhead-box protein P3 (Foxp3) have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC) the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC).
Methods and Findings: Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival.
Conclusions: Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression.
Molecular tumour targeting has significantly improved anti-cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti-tumour potential, but also to prevent resistance development seen under mono-drug therapy. This investigation was designed to evaluate whether cross-communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU-145 prostate cancer cells were treated with insulin-like growth factor (IGF) to activate the Akt-mTOR cascade or with the HDAC-inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock-down blocked the Akt-mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up-regulated histone acetylation, but also activated mTOR-Akt signalling. HDAC1 and 2 knock-down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC-mTOR communication, therefore, is apparent whereby tumour-promoting (Akt/mTORhigh, aH3/aH4low) and tumour-suppressing signals (Akt/mTORlow, aH3/aH4high) are activated in parallel. Combined use of an HDAC- and mTOR inhibitor might then diminish pro-tumour effects triggered by the HDAC- (Akt/mTORhigh) or mTOR inhibitor (aH3/aH4low) alone.