Methylation of GPRC5A promotes liver metastasis and docetaxel resistance through activating mTOR signaling pathway in triple negative breast cancer.

AIMS: This study aims to explore the function and mechanism of G Protein-coupled receptor class C group 5 member A (GPRC5A) in docetaxel-resistance and liver metastasis of breast cancer. METHODS: Single-cell RNA transcriptomic analysis and bioinformatic analysis are used to screen relevant genes in breast cancer metastatic hepatic specimens. MeRIP, dual-luciferase analysis and bioinformation were used to detect m6A modulation. Mass spectrometry (MS), co-inmunoprecipitation (co-IP) and immunofluorescence colocalization were executed to explore the mechanism of GPRC5A in breast cancer cells. RESULT: GPRC5A was upregulated in triple-negative breast cancer (TNBC) and was associated with a poor prognosis. In vitro and in vivo experiments demonstrated that knockdown of GPRC5A alleviated metastasis and resistance to docetaxel in TNBC. Overexpression of GPRC5A had the opposite effects. The m6A methylation of GPRC5A mRNA was modulated by METTL3 and YTHDF1, which facilitates its translation. GPRC5A inhibited the ubiquitination-dependent degradation of LAMTOR1, resulting in the recruitment of mTORC1 to lysosomes and activating the mTORC1/p70s6k signaling pathway. CONCLUSION: METTL3/YTHDF1 axis up-regulates GPRC5A expression by m6A methylation. GPRC5A activates mTORC1/p70s6k signaling pathway by recruiting mTORC1 to lysosomes, consequently promotes docetaxel-resistance and liver metastasis.

NOTCH3 promotes docetaxel resistance of prostate cancer cells through regulating TUBB3 and MAPK signaling pathway.

Docetaxel is the preferred chemotherapeutic agent in patients with castrate-resistant prostate cancer (CRPC). However, patients eventually develop docetaxel resistance and in the absence of effective treatment options. Consequently, it is essential to investigate the mechanisms generating docetaxel resistance and develop novel alternative therapeutic targets. RNA sequencing was undertaken on docetaxel-sensitive and docetaxel-resistant prostate cancer (PCa) cells. Subsequently, chemoresistance, cancer stemness, and lipid metabolism were investigated. To obtain insight into the precise activities and action mechanisms of NOTCH3 in docetaxel-resistant PCa, immunoprecipitation, mass spectrometry, ChIP, luciferase reporter assay, cell metabolism, and animal experiments were performed. Through RNA sequencing analysis, we found that NOTCH3 expression was markedly higher in docetaxel-resistant cells relative to parental cells, and that this trend was continued in docetaxel-resistant PCa tissues. Experiments in vitro and in vivo revealed that NOTCH3 enhanced stemness, lipid metabolism, and docetaxel resistance in PCa. Mechanistically, NOTCH3 is bound to TUBB3 and activates the MAPK signaling pathway. Moreover, NOTCH3 was directly regulated by MEF2A in docetaxel-resistant cells. Notably, targeting NOTCH3 and the MEF2A/TUBB3 signaling axis was related to docetaxel chemoresistance in PCa. Overall, these results demonstrated that NOTCH3 fostered stemness, lipid metabolism, and docetaxel resistance in PCa via the TUBB3 and MAPK signaling pathways. Therefore, NOTCH3 may be employed as a prognostic biomarker in PCa patients. NOTCH3 could be a therapeutic target for PCa patients, particularly those who have developed docetaxel resistance.

CircSLC4A7 in resistant-cells-derived exosomes promotes docetaxel resistance via the miR-1205/MAPT axis in prostate cancer.

Prostate cancer (PCa) is a high-mortality cancer. Docetaxel (DCT) combined with second-generation anti-androgens is considered the golden standard therapy for PCa, whose application is limited for DCT resistance (DR). Therefore, exploring the mechanism of DR is of great importance. In this study, PCa cell lines of PC3 and DU145 were employed, and DR cells were constructed by treatment with graded DCT. CircSLC4A7, miR-1205, and microtubule-associated protein tau (MAPT) transfections were established. Cell counting kit-8 assay was performed to evaluate the cell activity and IC50 of DCT. After being treated with DCT, DR was assessed by colony formation assay, flow cytometry analysis, and terminal transferase-mediated UTP nick end-labeling assay. Real-time quantitative PCR and western blotting analysis evaluated the expression levels of genes. The dual-luciferase reporter gene assay verified the miR-1205 binding sites with circSLC4A7 and MAPT. An animal experiment was performed to assess the tumor growth influenced by circSLC4A7. After conducting DR cells and isolated exosomes, we found that not only co-culture with DR cells but also treatment with DR cells' exosomes would promote the DR of normal cells. Moreover, circSLC4A7 was highly expressed in DR cells and their exosomes. CircSLC4A7 overexpression enhanced DR, represented as raised IC50 of DCT, increased colony formation, and decreased cell apoptosis after DCT treatment, while circSLC4A7 knockdown had the opposite effect. MiR-1205 was confirmed as a circSLC4A7-sponged miRNA and miR-1205 inhibitor reversed the effect of sh-circSLC4A7. MAPT was further identified as a target of miR-1205 and had a similar effect with circSLC4A7. The effect of circSLC4A7 on DR was also confirmed by xenograft experiments. Collectively, circSLC4A7 in resistant-cells-derived exosomes promotes DCT resistance of PCa via miR-1205/MAPT axis, which may provide a new treatment strategy for DR of PCa.

Epigenetic activation of METTL14 promotes docetaxel resistance in prostate cancer by promoting pri-microRNA-129 maturation.

The development of resistance to Docetaxel (DTX) compromises its therapeutic efficacy and worsens the prognosis of prostate cancer (PCa), while the underlying regulatory mechanism remains poorly understood. In this study, METTL14 was found to be upregulated in DTX-resistant PCa cells and PCa tissues exhibiting progressive disease during DTX therapy. Furthermore, overexpression of METTL14 promoted the development of resistance to DTX in both in vitro and in vivo. Interestingly, it was observed that the hypermethylation of the E2F1 targeting site within DTX-resistant PCa cells hindered the binding ability of E2F1 to the promoter region of METTL14, thereby augmenting its transcriptional activity. Consequently, this elevated expression level of METTL14 facilitated m6A-dependent processing of pri-miR-129 and subsequently led to an increase in miR-129-5p expression. Our study highlights the crucial role of the E2F1-METTL14-miR-129-5p axis in modulating DTX resistance in PCa, underscoring METTL14 as a promising therapeutic target for DTX-resistant PCa patients.

JMJD2A participates in cytoskeletal remodeling to regulate castration-resistant prostate cancer docetaxel resistance.

BACKGROUND: To investigate underlying mechanism of JMJD2A in regulating cytoskeleton remodeling in castration-resistant prostate cancer (CRPC) resistant to docetaxel. METHODS: Tissue samples from CRPC patients were collected, and the expression of JMJD2A, miR-34a and cytoskeleton remodeling-related proteins were evaluated by qPCR, western blot and immunohistochemistry, and pathological changes were observed by H&E staining. Further, JMJD2A, STMN1 and TUBB3 were knocked down using shRNA in CRPC cell lines, and cell viability, apoptosis and western blot assays were performed. The interaction between miR-34a/STMN1/ß3-Tubulin was analyzed with dual-luciferase reporter and co-immunoprecipitation assays. RESULTS: In clinical experiment, the CRPC-resistant group showed higher expression of JMJD2A, STMN1, α-Tubulin, ß-Tubulin and F-actin, and lower expression of miR-34a and ß3-Tubulin compared to the sensitive group. In vitro experiments showed that JMJD2A could regulate cytoskeletal remodeling through the miR-34a/STMN1/ß3-Tubulin axis. The expression of miR-34a was elevated after knocking down JMJD2A, and miR-34a targeted STMN1. The overexpression of miR-34a was associated with a decreased expression of STMN1 and elevated expression of ß3-Tubulin, which led to the disruption of the microtubule network, decreased cancer cell proliferation, cell cycle arrest in the G0/G1 phase, and increased apoptosis. CONCLUSION: JMJD2A promoted docetaxel resistance in prostate cancer cells by regulating cytoskeleton remodeling through the miR-34a/STMN1/ß3-Tubulin axis.

Synthesis and biological evaluation of atropisomeric tetrahydroisoquinolines overcoming docetaxel resistance in triple-negative human breast cancer cells.

Herein, atropisomeric 8-aryltetrahydroisoquinolines have been synthesized and biologically evaluated. Based on our structure-activity relationship study, a highly bioactive racemic compound has been produced, and it exhibited high antiproliferative activities against various cancer cell lines, including docetaxel-resistant breast cancer cell lines. Each enantiomer can be synthesized in an enantioselective manner by employing the chiral phosphoric acid-catalyzed atroposelective Pictet-Spengler cyclization. An axially (R)-configured enantiomer showed a higher biological activity compared with the axially (S)-configured enantiomer. Further biological studies suggested that the (R)-enantiomer overcomes docetaxel resistance via the downregulation of signal transducer and activator of transcription 3 activation and consequently induces cellular apoptosis in docetaxel-resistant triple-negative breast cancer cell lines.

CircRNA CircABCB1 Diminishes the Sensitivity of Breast Cancer Cells to Docetaxel by Sponging MiR-153-3p.

Resistance to docetaxel is a major problem to the success of docetaxel-based therapies for breast cancer. The present study was to identify the role of circABCB1 in altering the docetaxel resistance properties. Reverse transcription-quantitative PCR (qRT-PCR) was performed to quantify circABCB1 and miR-153-3p. The effects of circABCB1 on the viability, apoptosis and migration/invasion of docetaxel-resistant and -sensitive cells were investigated by cell function experiments, including Cell Counting Kit-8 and Transwell assays. Correlation between circABCB1 and the docetaxel-treated outcome was analyzed by multivariate Cox regression analysis, in addition to Kaplan-Meier analysis of time to treatment failure (TTF). The targeting relationship between circABCB1 and miR-153-3p was predicted and verified by dual-luciferase reporter assay and RNA immunoprecipitation. CircABCB1 was highly expressed in cancerous tissues, as well as the docetaxel-sensitive group and cells. The overexpression of circABCB1 contributed to cell viability, docetaxel-resistance and migration/invasion, but inhibited apoptosis. CircABCB1 can sponge miR-153-3p. CircABCB1 contributed to the docetaxel resistance of breast cancer, maybe via the miR-153-3p.

Comparative proteome analysis identified CD44 as a possible serum marker for docetaxel resistance in castration-resistant prostate cancer.

Baseline or acquired resistance to docetaxel (DOC) represents a significant risk for patients with metastatic prostate cancer (PC). In the last years, novel therapy regimens have been approved providing reasonable alternatives for DOC-resistant patients making prediction of DOC resistance of great clinical importance. We aimed to identify serum biomarkers, which are able to select patients who will not benefit from DOC treatment. DOC-resistant PC3-DR and DU145-DR sublines and their sensitive parental cell lines (DU145, PC3) were comparatively analyzed using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Results were filtered using bioinformatics approaches to identify promising serum biomarkers. Serum levels of five proteins were determined in serum samples of 66 DOC-treated metastatic castration-resistant PC patients (mCRPC) using ELISA. Results were correlated with clinicopathological and survival data. CD44 was subjected to further functional cell culture analyses. We found at least 177 two-fold significantly overexpressed proteins in DOC-resistant cell lines. Our bioinformatics method suggested 11/177 proteins to be secreted into the serum. We determined serum levels of five (CD44, MET, GSN, IL13RA2 and LNPEP) proteins in serum samples of DOC-treated patients and found high CD44 serum levels to be independently associated with poor overall survival (p = 0.001). In accordance, silencing of CD44 in DU145-DR cells resulted in re-sensitization to DOC. In conclusion, high serum CD44 levels may help identify DOC-resistant patients and may thereby help optimize clinical decision-making regarding type and timing of therapy for mCRPC patients. In addition, our in vitro results imply the possible functional involvement of CD44 in DOC resistance.

MARCKSL1-2 reverses docetaxel-resistance of lung adenocarcinoma cells by recruiting SUZ12 to suppress HDAC1 and elevate miR-200b.

BACKGROUND: Long non-coding RNAs (lncRNAs) are implicated in the development of multiple cancers. In our previous study, we demonstrated that HDAC1/4-mediated silencing of microRNA-200b (miR-200b) enhances docetaxel (DTX)-resistance of human lung adenocarcinoma (LAD) cells. METHODS AND RESULTS: Herein, we probed the function of LncRNA MARCKSL1-2 (MARCKSL1-transcript variant 2, NR_052852.1) in DTX resistance of LAD cells. It was found that MARCKSL1-2 expression was markedly reduced in DTX-resistant LAD cells. Through gain- or loss- of function assays, colony formation assay, EdU assay, TUNEL assay, and flow cytometry analysis, we found that MARCKSL1-2 suppressed the growth and DTX resistance of both parental and DTX-resistant LAD cells. Moreover, we found that MARCKSL1-2 functioned in LAD through increasing miR-200b expression and repressing HDAC1. Mechanistically, MARCKSL1-2 recruited the suppressor of zeste 12 (SUZ12) to the promoter of histone deacetylase 1 (HDAC1) to strengthen histone H3 lysine 27 trimethylation (H3K27me3) of HDAC1 promoter, thereby reducing HDAC1 expression. MARCKSL1-2 up-regulated miR-200b by blocking the suppressive effect of HDAC1 on the histone acetylation modification at miR-200b promoter. Furthermore, in vivo analysis using mouse xenograft tumor model supported that overexpression of MARCKSL1-2 attenuated the DTX resistance in LAD tumors. CONCLUSIONS: We confirmed that MARCKSL1-2 alleviated DTX resistance in LAD cells by abolishing the inhibitory effect of HDAC1 on miR-200b via the recruitment of SUZ12. MARCKSL1-2 could be a promising target to improve the chemotherapy of LAD.

miR-1205/DNAJB1 reverses docetaxel chemoresistance in human triple negative breast carcinoma cells via regulation of mutp53/TAp63 signaling.

Chemoresistance is the major cause of therapeutic failure in human triple negative breast carcinoma (TNBC). Docetaxel (DOC), a first-line therapeutic drug in TNBC treatment, is limited for long-term use due to the development of chemoresistance. Thus, overcoming chemoresistance of DOC remains an important challenge to improve patient's outcome of TNBC. In this study, we aimed to investigate the molecular mechanism behind DOC chemoresistance and the possible therapeutic effects of miRNAs. Utilizing qRT-PCR analysis, we discovered that miR-1205 is gradually downregulated in human triple negative breast carcinoma MDA-MB-231 and docetaxel-resistant MDA-MB-231 (MDA-MB-231/DOC) cells compared with Hs 578Bst normal human breast fibroblasts. Cell viability, cell cycle and apoptosis assays in MDA-MB-231/DOC cells indicated that miR-1205 overexpression enhances docetaxel sensitivity by reducing cell viability as well as inducing G2/M cell cycle arrest and cell apoptosis. Western blot analysis, dual-luciferase reporter assay, co-immunoprecipitation assay and chromatin immunoprecipitation assay revealed that miR-1205 overexpression disrupts the stable complex formation of DNAJB1, mutp53 and TAp63 by directly reducing DNAJB1 expression, which abates the sequestrating effect of mutp53 on TAp63, thereby leading to the enhanced DOC sensitivity in MDA-MB-231/DOC cells. Our findings demonstrate the role of the miR-1205/DNAJB1 axis in the docetaxel resistance of TNBC, which may offer a promising therapeutic approach to resolve docetaxel resistance in TNBC.

Hsa_circ_0006404 and hsa_circ_0000735 Regulated Ovarian Cancer Response to Docetaxel Treatment via Regulating p-GP Expression.

Several microRNAs (miRNAs) and circular RNAs (circRNAs) were reported to be involved in the Docetaxel (DTX) chemoresistance of cancer treatment, but the underlying mechanisms remain to be explored. In this study, we established cellular and animal models respectively to study the effect and underlying molecular mechanisms of the dysregulation of circRNA_0006404 and circRNA_0000735 in tumor response to DTX treatment. Quantitative real-time PCR was performed to measure the expression of circRNA_0006404, miR-346, circRNA_0000735, miR-526b, Dickkopf-related protein 3 (DKK3), and Dickkopf-related protein 4 (DKK4) mRNA. The expression of circRNA_0006404 and circRNA_0000735 was remarkably suppressed and activated in DTX-treated SKOV3-R cell lines, respectively. As revealed by luciferase assays, circRNA_0006404 and circRNA_0000735 was found to be respectively targeted by miR-346 and miR-526b, while DKK3 and DKK4 were respectively targeted by miR-346 and miR-526b. Moreover, the expression of DKK3 and DKK4, which were targets of miR-346 and miR-526b, respectively, was significantly altered along with the expression of p-GP. Furthermore, circ_0006404 shRNA and circRNA_0000735 shRNA showed remarkable efficiency in stimulating the expression of circRNA_0006404, miR-346, DKK3, circRNA_0000735, miR-526b, DKK4, and p-GP in cellular and animal models. Accordingly, the cell apoptosis and proliferation were apparently changed by circ_0006404 shRNA and circRNA_0000735 shRNA in both cellular and animal models. In summary, our study found the involvement of the circRNA_0006404/miR-346/DKK3/p-GP and circRNA_0000735/miR-546b/DKK4/p-GP axis in the tumor response to DTX. Both the up-regulation of circRNA_0006404 and down-regulation of circRNA_0000735 could inhibit the expression of p-GP in vivo and ex vivo, leading to the suppressed tumor response to DTX treatment.

Transcription factor network analysis based on single cell RNA-seq identifies that Trichostatin-a reverses docetaxel resistance in prostate Cancer.

BACKGROUND: Overcoming drug resistance is critical for increasing the survival rate of prostate cancer (PCa). Docetaxel is the first cytotoxic chemotherapeutical approved for treatment of PCa. However, 99% of PCa patients will develop resistance to docetaxel within 3 years. Understanding how resistance arises is important to increasing PCa survival. METHODS: In this study, we modeled docetaxel resistance using two PCa cell lines: DU145 and PC3. Using the Passing Attributes between Networks for Data Assimilation (PANDA) method to model transcription factor (TF) activity networks in both sensitive and resistant variants of the two cell lines. We identified edges and nodes shared by both PCa cell lines that composed a shared TF network that modeled changes which occur during acquisition of docetaxel resistance in PCa. We subjected the shared TF network to connectivity map analysis (CMAP) to identify potential drugs that could disrupt the resistant networks. We validated the candidate drug in combination with docetaxel to treat docetaxel-resistant PCa in both in vitro and in vivo models. RESULTS: In the final shared TF network, 10 TF nodes were identified as the main nodes for the development of docetaxel resistance. CMAP analysis of the shared TF network identified trichostatin A (TSA) as a candidate adjuvant to reverse docetaxel resistance. In cell lines, the addition of TSA to docetaxel enhanced cytotoxicity of docetaxel resistant PCa cells with an associated reduction of the IC50 of docetaxel on the resistant cells. In the PCa mouse model, combination of TSA and docetaxel reduced tumor growth and final weight greater than either drug alone or vehicle. CONCLUSIONS: We identified a shared TF activity network that drives docetaxel resistance in PCa. We also demonstrated a novel combination therapy to overcome this resistance. This study highlights the usage of novel application of single cell RNA-sequencing and subsequent network analyses that can reveal novel insights which have the potential to improve clinical outcomes.

MiR-129-5p promotes docetaxel resistance in prostate cancer by down-regulating CAMK2N1 expression.

This study focuses on the effect of miR-129-5p on docetaxel-resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT-PCR in PCa patient tissues and cell lines including PC-3 and PC-3-DR. Cells transfected with miR-129-5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR-129-5p and CAMK2N1 levels were identified by qRT-PCR and dual-luciferase reporter assay. CAMK2N1 was found to be down-expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up-regulation of miR-129-5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR-129-5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR-129-5p contributed to the resistance of PC-3-DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR-129-5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.

Acetyl-11-keto-ß-boswellic acid suppresses docetaxel-resistant prostate cancer cells in vitro and in vivo by blocking Akt and Stat3 signaling, thus suppressing chemoresistant stem cell-like properties.

Acquired docetaxel-resistance of prostate cancer (PCa) remains a clinical obstacle due to the lack of effective therapies. Acetyl-11-keto-ß-boswellic acid (AKBA) is a pentacyclic triterpenic acid isolated from the fragrant gum resin of the Boswellia serrata tree, which has shown intriguing antitumor activity against human cell lines established from PCa, colon cancer, malignant glioma, and leukemia. In this study, we examined the effects of AKBA against docetaxel-resistant PCa in vitro and in vivo as well as its anticancer mechanisms. We showed that AKBA dose-dependently inhibited cell proliferation and induced cell apoptosis in docetaxel-resistant PC3/Doc cells; its IC50 value in anti-proliferation was ∼17 µM. Furthermore, AKBA dose-dependently suppressed the chemoresistant stem cell-like properties of PC3/Doc cells, evidenced by significant decrease in the ability of mammosphere formation and down-regulated expression of a number of stemness-associated genes. The activation of Akt and Stat3 signaling pathways was remarkably enhanced in PC3/Doc cells, which contributed to their chemoresistant stem-like phenotype. AKBA (10-30 µM) dose-dependently suppressed the activation of Akt and Stat3 signaling pathways in PC3/Doc cells. In contrast, overexpression of Akt and Stat3 significantly attenuated the inhibition of AKBA on PC3/Doc cell proliferation. In docetaxel-resistant PCa homograft mice, treatment with AKBA significantly suppresses the growth of homograft RM-1/Doc, equivalent to its human PC3/Doc, but did not decrease their body weight. In summary, we demonstrate that AKBA inhibits the growth inhibition of docetaxel-resistant PCa cells in vitro and in vivo via blocking Akt and Stat3 signaling, thus suppressing their cancer stem cell-like properties.

The prognostic utility of the transcription factor SRF in docetaxel-resistant prostate cancer: in-vitro discovery and in-vivo validation.

BACKGROUND: Docetaxel based therapy is one of the first line chemotherapeutic agents for the treatment of metastatic castrate-resistant prostate cancer. However, one of the major obstacles in the treatment of these patients is docetaxel-resistance. Defining the mechanisms of resistance so as to inform subsequent treatment options and combinations represents a challenge for clinicians and scientists. Previous work by our group has shown complex changes in pro and anti-apoptotic proteins in the development of resistance to docetaxel. Targeting these changes individually does not significantly impact on the resistant phenotype but understanding the central signalling pathways and transcription factors (TFs) which control these could represent a more appropriate therapeutic targeting approach. METHODS: Using a number of docetaxel-resistant sublines of PC-3 cells, we have undertaken a transcriptomic analysis by expression microarray using the Affymetrix Human Gene 1.0 ST Array and in conjunction with bioinformatic analyses undertook to predict dysregulated TFs in docetaxel resistant prostate cancer. The clinical significance of this prediction was ascertained by performing immunohistochemical (IHC) analysis of an identified TF (SRF) in the metastatic sites from men who died of advanced CRPC. Investigation of the functional role of SRF was examined by manipulating SRF using SiRNA in a docetaxel-resistant PC-3 cell line model. RESULTS: The transcription factors identified include serum response factor (SRF), nuclear factor kappa-B (NFκB), heat shock factor protein 1 (HSF1), testicular receptor 2 & 4 (TR2 &4), vitamin-D and retinoid x receptor (VDR-RXR) and oestrogen-receptor 1 (ESR1), which are predicted to be responsible for the differential gene expression observed in docetaxel-resistance. IHC analysis to quantify nuclear expression of the identified TF SRF correlates with both survival from date of bone metastasis (p = 0.003), survival from androgen independence (p = 0.00002), and overall survival from prostate cancer (p = 0.0044). Functional knockdown of SRF by siRNA demonstrated a reversal of apoptotic resistance to docetaxel treatment in the docetaxel-resistant PC-3 cell line model. CONCLUSIONS: Our results suggest that SRF could aid in treatment stratification of prostate cancer, and may also represent a therapeutic target in the treatment of men afflicted with advanced prostate cancer.

The Prostate Cancer Cells Resistant to Docetaxel as in vitro Model for Discovering MicroRNAs Predictive of the Onset of Docetaxel Resistance.

On the grounds that miRNAs present in the blood of prostate cancer (PCa) patients are released in the growth medium by PCa cells, it is conceivable that PCa cells resistant to docetaxel (DCT) (DCTR) will release miRNAs that may be found in PCa patients under DCT therapy if resistant PCa cells appear. We isolated DCTR clones respectively from 22Rv1 and DU-145 PCa cell lines and performed through next-generation sequencing (NGS) the miRNAs profiles of the released miRNAs. The analysis of the NGS data identified 105 and 1 miRNAs which were differentially released in the growth medium of the 22Rv1/DCTR and DU-145/DCTR clones, respectively. Using additional filters, we selected 12 and 1 miRNA more released by all 22Rv1/DCTR and DU-145/DCTR clones, respectively. Moreover, we showed that 6 of them were more represented in the growth medium of the DCTR cells than the ones of DCT-treated cells. We speculated that they have the pre-requisite to be tested as predictive biomarkers of the DCT resistance in PCa patients under DCT therapy. We propose the utilization of clones resistant to a given drug as in vitro model to identify the differentially released miRNAs, which in perspective could be tested as predictive biomarkers of drug resistance in tumor patients under therapy.

The stepwise evolution of the exome during acquisition of docetaxel resistance in breast cancer cells.

BACKGROUND: Resistance to taxane-based therapy in breast cancer patients is a major clinical problem that may be addressed through insight of the genomic alterations leading to taxane resistance in breast cancer cells. In the current study we used whole exome sequencing to discover somatic genomic alterations, evolving across evolutionary stages during the acquisition of docetaxel resistance in breast cancer cell lines. RESULTS: Two human breast cancer in vitro models (MCF-7 and MDA-MB-231) of the step-wise acquisition of docetaxel resistance were developed by exposing cells to 18 gradually increasing concentrations of docetaxel. Whole exome sequencing performed at five successive stages during this process was used to identify single point mutational events, insertions/deletions and copy number alterations associated with the acquisition of docetaxel resistance. Acquired coding variation undergoing positive selection and harboring characteristics likely to be functional were further prioritized using network-based approaches. A number of genomic changes were found to be undergoing evolutionary selection, some of which were likely to be functional. Of the five stages of progression toward resistance, most resistance relevant genomic variation appeared to arise midway towards fully resistant cells corresponding to passage 31 (5 nM docetaxel) for MDA-MB-231 and passage 16 (1.2 nM docetaxel) for MCF-7, and where the cells also exhibited a period of reduced growth rate or arrest, respectively. MCF-7 cell acquired several copy number gains on chromosome 7, including ABC transporter genes, including ABCB1 and ABCB4, as well as DMTF1, CLDN12, CROT, and SRI. For MDA-MB-231 numerous copy number losses on chromosome X involving more than 30 genes was observed. Of these genes, CASK, POLA1, PRDX4, MED14 and PIGA were highly prioritized by the applied network-based gene ranking approach. At higher docetaxel concentration MCF-7 subclones exhibited a copy number loss in E2F4, and the gene encoding this important transcription factor was down-regulated in MCF-7 resistant cells. CONCLUSIONS: Our study of the evolution of acquired docetaxel resistance identified several genomic changes that might explain development of docetaxel resistance. Interestingly, the most relevant resistance-associated changes appeared to originate midway through the evolution towards fully resistant cell lines. Our data suggest that no single genomic event sufficiently predicts resistance to docetaxel, but require genomic alterations affecting multiple pathways that in concert establish the final resistance stage.

miR-375 induces docetaxel resistance in prostate cancer by targeting SEC23A and YAP1.

BACKGROUND: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically are centered on docetaxel-based chemotherapy. We previously reported that elevated miR-375 levels were significantly associated with poor overall survival of mCRPC patients. In this study, we evaluated if miR-375 induced chemo-resistance to docetaxel through regulating target genes associated with drug resistance. METHODS: We first compared miR-375 expression level between prostate cancer tissues and normal prostate tissues using data from The Cancer Genome Atlas (TCGA). To examine the role of miR-375 in docetaxel resistance, we transfected miR-375 using a pre-miRNA lentiviral vector and examined the effects of exogenously overexpressed miR-375 on cell growth in two prostate cancer cell lines, DU145 and PC-3. To determine the effect of overexpressed miR-375 on tumor growth and chemo-resistance in vivo, we injected prostate cancer cells overexpressing miR-375 into nude mice subcutaneously and evaluated tumor growth rate during docetaxel treatment. Lastly, we utilized qRT-PCR and Western blot assay to examine two miR-375 target genes, SEC23A and YAP1, for their expression changes after miR-375 transfection. RESULTS: By examining 495 tumor tissues and 52 normal tissues from TCGA data, we found that compared to normal prostate, miR-375 was significantly overexpressed in prostate cancer tissues (8.45-fold increase, p value = 1.98E-23). Docetaxel treatment induced higher expression of miR-375 with 5.83- and 3.02-fold increases in DU145 and PC-3 cells, respectively. Interestingly, miR-375 appeared to play a dual role in prostate cancer proliferation. While miR-375 overexpression caused cell growth inhibition and cell apoptosis, elevated miR-375 also significantly reduced cell sensitivity to docetaxel treatment in vitro, as evidenced by decreased apoptotic cells. In vivo xenograft mouse study showed that tumors with increased miR-375 expression were more tolerant to docetaxel treatment, demonstrated by greater tumor weight and less apoptotic cells in miR-375 transfected group when compared to empty vector control group. In addition, we examined expression levels of the two miR-375 target genes (SEC23A and YAP1) and observed significant reduction in the expression at both protein and mRNA levels in miR-375 transfected prostate cancer cell lines. TCGA dataset analysis further confirmed the negative correlations between miR-375 and the two target genes (r = -0.62 and -0.56 for SEC23A and YAP1, respectively; p < 0.0001). CONCLUSIONS: miR-375 is involved in development of chemo-resistance to docetaxel through regulating SEC23A and YAP1 expression. Our results suggest that miR-375 or its target genes, SEC23A or YAP1, might serve as potential predictive biomarkers to docetaxel-based chemotherapy and/or therapeutic targets to overcome chemo-resistance in mCRPC stage.

The presence and clinical implication of intraductal carcinoma of prostate in metastatic castration resistant prostate cancer.

BACKGROUND: Intraductal carcinoma of prostate (IDC-P) is always underestimated pathological pattern in prostate cancer and its role is still unclear in castration resistant prostate cancer (CRPC). This study was conducted to investigate the presence and the roles of IDC-P in patients with metastatic CRPC. METHODS: 45 patients with initially diagnosed metastatic prostate cancer and then progressed to CRPC, were included. All of them were received twice transperineal biopsies at the time of initial diagnosis and the time of CRPC. All samples were retrieved to detect the presence of IDC-P. PSA doubling time (PSADT) was considered as a parameter presenting the progression of CRPC. The relationships between IDC-P and other clinicopathological variables were analyzed. RESULTS: IDC-P was found only in 20% (9/45) cases at initial diagnosis, whereas, it increased to 62.5% (28/45) at the time of CRPC (χ(2) = 16.568, P = 0.000). Compared to acinar adenocarcinoma components in tumor tissues, IDC-P components, especially solid subtype, had obviously poor/no response to androgen deprivation therapy (ADT). In addition, among patients treated with docetaxel-based chemotherapy (n = 24), patients with IDC-P also showed more unfavorable response than those without IDC-P (20% vs. 66.7%, P = 0.022). The presence of IDC-P and serum testosterone at the time of CRPC, were significantly associated with rapid disease progression. 13/28 (46.4%) CRPC with IDC-P had PSADT less than 30 days, while, only 1/17 (5.9%) patient without IDC-P had a less than 30 days PSADT (χ(2) = 8.114, P = 0.004). Limitations included the relative short follow-up time and a relative small cohort. CONCLUSIONS: The presence of IDC-P was significantly associated with rapid progression of CRPC. And its presence could suggest the poor response to initial ADT and sequential docetaxel-based chemotherapy. Detection of IDC-P should be of importance in CRPC, and re-biopsy at the time of CRPC might be one of practical solutions. The mechanism of the ADT and docetaxel resistance to IDC-P needed to be further investigated.

FOXM1 mediates resistance to docetaxel in gastric cancer via up-regulating Stathmin.

Docetaxel is commonly used as an effective chemotherapeutic drug for gastric cancer patients recently. With the increasing emergence of docetaxel resistance nowadays, identification of suitable biomarkers for predicting chemosensitivity to docetaxel may be a key role for improving therapeutic effects for gastric cancer patients. In this study, we investigated the correlation between the expression of transcription factor forkhead box protein M1 (FOXM1) and chemotherapy response to docetaxel in gastric cancer, the possible mechanism for which was further explored. As a result, FOXM1 overexpression was shown to mediate resistance to docetaxel in gastric cancers. It altered microtubule dynamics to protect tumour cells from docetaxel-induced apoptosis. Mechanistic investigations revealed that tubulin-destabilizing protein Stathmin, which mediated docetaxel resistance in FOXM1-silenced gastric cancer cells, is a direct down-stream target of FOXM1, whereas another microtubule dynamics protein mitotic centromere-associated kinesin (MCAK), shown to be related to docetaxel resistance in gastric cancer cells, is not associated with FOXM1 expression significantly. These results were further provided by immunohistochemical analysis, indicating that FOXM1 and Stathmin expression levels were correlated in 103 post-operational gastric cancer specimens. Moreover, when we attenuated FOXM1 expression with FOXM1 inhibitor thiostrepton, docetaxel resistance in gastric cancers was found to be reversed, simultaneously with the down-regulation of FOXM1 and Stathmin. Therefore, FOXM1 can be a useful marker for predicting and monitoring docetaxel response. Through the inhibition of FOXM1, docetaxel resistance can be reversed, and thus FOXM1 could be a new therapeutic target in docetaxel-resistant gastric cancer.