Downregulation of miR-99b-5p and Upregulation of Nuclear mTOR Cooperatively Promotes the Tumor Aggressiveness and Drug Resistance in African American Prostate Cancer

Abstract

Mammalian target of rapamycin (mTOR) regulates various fundamental cellular events including cell proliferation, protein synthesis, metabolism, apoptosis, and autophagy. Tumor suppressive miR-99b-5p has been implicated in regulating PI3K/AKT/mTOR signaling in a variety of types of cancer. Our previous study suggested the reciprocal miR-99b-5p/MTOR (downregulated/upregulated) pairing as a key microRNA-mRNA regulatory component involved in the prostate cancer (PCa) disparities. In this study, we further validated the expression profiles of mTOR and miR-99b-5p in the PCa, colon, breast, and lung cancer specimens and cell lines. The immunohistochemistry (IHC), immunofluorescence, Western blot, and RT-qPCR assays have confirmed that mTOR is upregulated while miR-99b-5p is downregulated in different patient cohorts and a panel of cancer cell lines. Intriguingly, elevated nuclear mTOR expression was observed in African American PCa and other advanced cancers. Transfection of the miR-99b-5p mimic resulted in a significant reduction in nuclear mTOR and androgen receptor (AR), while a slight/moderate to no decrease in cytoplasmic mTOR and AR in PCa and other cancer cells, suggesting that miR-99b-5p inhibits mTOR and AR expression and their nuclear translocation. Moreover, overexpression of miR-99b-5p targets/inhibits AR-mTOR axis, subsequently initiating cell apoptosis and sensitizing docetaxel-induced cytotoxicity in various cancers. In conclusion, our data suggest that reciprocal miR-99b-5p/nuclear mTOR pairing may be a more precise diagnostic/prognostic biomarker for aggressive PCa, than miR-99b-5p/MTOR pairing or mTOR alone. Targeting the AR-mTOR axis using miR-99b-5p has also been suggested as a novel therapeutic strategy to induce apoptosis and overcome chemoresistance in aggressive PCa.

Keywords: miR-99b-5p; nuclear pmTOR; precision prognostic biomarker; reciprocal miRNA-mRNA pairing.

Figure 1

IHC staining assays for examining mTOR and AMACR protein levels in PCa patient specimens. (A) Quantification of IHC staining signals from mTOR in EA and AA PCa specimens (left panel) and representative IHC images from EA and AA PCa specimens (right panel). NEA: adjacent normal specimen derived from EA patient, TEA: tumorous EA specimen, NAA: adjacent normal specimen from AA PCa, TEA: tumorous AA specimen. Higher mTOR intensities were shown in AA PCa vs. EA PCa with comparable Gleason scores (3 + 4, 4 + 4, and 5 + 4). *** Significance (p-value < 0.001, comparing mTOR staining intensities in AA PCa vs. EA PCa specimens) was determined based on ANOVA with Tukey post-hoc test. (B) Quantification (left panel) and representative IHC images of mTOR and AMACR staining in PCa patient specimens (right panel). mTOR and AMACR staining intensities were measured in normal prostate tissues and PCa specimens in the TMAs. GS: Gleason Score. **** Significance (p-value < 0.0001, comparing AMACR or mTOR staining intensities in PCa vs. normal tissues) was determined based on paired t-test. (C) Higher frequency of nuclear mTOR signals was detected in AA PCa specimens (left panel). Representative IHC staining revealed both nuclear (Nuc) and cytoplasmic (Cy) mTOR expression in EA PCa (TEA) and AA PCa (TAA) specimens. Note that high-level nuclear (nearly exclusive) mTOR signals were detected in TAA #2 and #3 samples (right panel). Percentage of nuclear mTOR-positive TEA and TAA samples were calculated based on the equation of (number of nuclear mTOR-positive specimens/number of mTOR-positive specimens) × 100% in TEAs and TAAs, respectively.

Figure 2

Immunofluorescence staining demonstrated higher expression levels of mTOR, nuclear mTOR, and nuclear pmTOR in AA PCa when compared to EA PCa cells. (A) Immunofluorescence showing mTOR (green fluorescence) and pmTOR (red fluorescence) signals in EA PCa cell lines (22Rv1, LNCaP and PC-3), and AA PCa cell lines (RC77 T/E and MDA PCa 2b). Nuclei were visualized by counterstaining with DAPI (blue fluorescence). Merged images were achieved by overlaying DAPI, mTOR and pmTOR signals to identify colocalization (yellow) of mTOR and pmTOR in nuclei. Fluorescent image capturing and analysis were performed using CellScans software V1.18. (B) Total mTOR and nuclear mTOR signals in EA and AA PCa cell lines. Fluorescence mTOR- or pmTOR-positive cells (%) were determined based on (number of mTOR or pmTOR-positive cells/number of DAPI-positive cells) × 100%. Significance was determined based on ANOVA with Tukey’s post-hoc test (* p-value < 0.001 in 22Rv1 vs. RC77 T/E or vs. MDA PCa 2b, ** p-value < 0.001 in LNCaP vs. RC77 T/E or vs. MDA PCa 2b, and *** p-value < 0.001 in PC-3 vs. RC77 T/E or vs. MDA PCa 2b). (C) Distribution of cytoplasmic and nuclear pmTOR in EA and AA PCa cell lines. Significant difference of nuclear mTOR (* p-value < 0.001 in 22Rv1 vs. RC77 T/E or vs. MDA PCa 2b, ** p-value < 0.001 in LNCaP vs. RC77 T/E or vs. MDA PCa 2b, and *** p-value < 0.001 in PC-3 vs. RC77 T/E or vs. MDA PCa 2b) and nuclear pmTOR (^# p-value < 0.001 in 22Rv1 vs. RC77 T/E or vs. MDA PCa 2b, ^## p-value < 0.001 in LNCaP vs. RC77 T/E or vs. MDA PCa 2b, and ^### p-value < 0.001 in PC-3 vs. RC77 T/E or vs. MDA PCa 2b) were shown in AA PCa vs. EA PCa. The statistics were determined based on ANOVA with Tukey’s post-hoc test, and each value was represented as mean ± SEM (n = 6).

Figure 3

Transfection of miR-99b-5p mimic attenuates mTOR and pmTOR expressions and blocks the translocation of pmTOR to the nuclei. (A) Immunofluorescence showing cytoplasmic and nuclear localizations of mTOR (green fluorescence) and pmTOR (red fluorescence) in EA PCa cell lines (22Rv1, LNCaP and PC-3) and AA PCa cell lines (RC77 T/E and MDA PCa 2b) transfected with NS or miR-99b-5p mimic. The nuclei were visualized by counterstaining with DAPI. (B) Quantification analysis of cytoplasmic and nuclear distribution in EA and AA PCa cells. Significant increase in cytoplasmic pmTOR (^# p-value < 0.01 using ANOVA with Tukey’s post-hoc test) and a significant decrease in nuclear pmTOR (* p-value using ANOVA with Tukey’s post-hoc test) was observed in the miR-99b-5p mimic vs. NS transfected cells. Each value was represented as mean ± SEM (n = 6). (C) Western blot analysis of mTOR and pmTOR in cytoplasmic and nuclear fractions of EA and AA PCa cell lines transfected with NS or the miR-99b-5p mimic. The representative images shown here were selected from 3–4 independent Western blot results. GAPDH and Lamin B1 were used as endogenous controls for cytoplasmic (Cy) and nuclear (Nu) proteins, respectively.

Figure 4

mTOR and miR-99b-5p expression profiles in colon, breast, and lung cancer cell models. (A) Quantification of mTOR intensities in colon, breast, lung, and prostate cancer specimens using IHC staining assay. IHC staining assay was applied to examine mTOR protein levels in various solid tumor patient specimens on a TMA slide. Significantly different mTOR intensities were identified in PCa vs. breast cancer specimens (**** p-value < 0.0001, based on ANOVA with Dunnett’s post-hoc test). No significant difference (ns) in mTOR intensities was found in colon cancer vs. PCa, or lung cancer vs. PCa specimens. (B) Representative IHC staining images of the mTOR protein in colon, breast, and lung cancer specimens. Apparently, the expression levels of mTOR were gradually increased from low- to high-grade cancer samples. G1: grade 1 tumor, G2: grade 2 tumor, and G3: grade 3 tumor. (C) Western blot analysis of mTOR protein levels in a panel of normal and cancer cell lines. Representative Western blot analysis of mTOR protein levels from total protein extracts of colon cancer (HT-29, SW620), breast cancer (MDA MB 231, MCF-7), lung cancer (A549, H1299), and PCa (PC-3, MDA PCa 2b) cell lines and control cell lines (FHC, HMEC and BEAS-2B from normal colon, breast and lung tissues, respectively). (D) RT-qPCR assays showed downregulation of miR-99b-5p in colon, breast and lung cancer cell lines, compared to their normal controls. RNA samples isolated from FHC, HMEC, HT-29, SW620, MDA MB 231, MCF-7, BEAS-2B, A549, and H1299 were subjected to RT-qPCR assays of miR-99b-5p. Significantly different miR-99b-5p expression levels (**** p-value < 0.0001 and *** p-value < 0.001, based on ANOVA with Tukey’s post-hoc test) were shown in cancer cell lines vs. normal controls (except A549 vs. BEAS-2B). ns: not significant. Each value was represented as mean ± SEM, obtained from three independent cDNA samples with duplicate or triplicate qPCR reactions. ns: not significant.

Figure 5

Overexpression of miR-99b-5p changes the subcellular distribution of mTOR and pmTOR and initiates cell apoptosis. (A) Immunofluorescence assays were used to visualize the subcellular localization of mTOR (green fluorescence) and pmTOR (green fluorescence) signals in cancer cell lines (HT-29, SW620, MDA MB 231, MCF-7, A549, and H1299) transfected with NS or miR-99b-5p mimic. Nuclei were visualized by counterstaining with DAPI. (B) TUNEL assays were used to visualize the DNA damages created during apoptotic events in the cancer cell lines upon miR-99b-5p and NS transfections. Apoptotic events were detected based on the DNA damages (visualized as red fluorescent spot signals, TUNEL panel) in the nuclei (blue, DAPI panel). The red/purple signals shown by overlaying DAPI and TUNEL signals (Merge panel) indicated apoptotic activities (DNA damages occurring in the nuclei) in the cancer cells.

Figure 6

Overexpression of miR-99b-5p inhibits mTOR and pmTOR expression and nuclear translocation, and sensitizes the docetaxel-induced cytotoxicity in cancer cells. Western blot analyses were used to examine: (A) mTOR and pmTOR protein levels in cytoplasmic and nuclear fractions. GAPDH and Lamin B1 were used as endogenous controls for cytoplasmic (Cy) and nuclear (Nu) proteins, respectively. (B) AR levels in total cell lysates (Total), cytoplasm (Cy), and nuclei (Nu) from the cancer cells (MCF-7, 22Rv1, and MDA PCa 2b) transfected with NS or miR-99b-5p mimic. The representative images were selected from 3–4 independent Western blot results. GAPDH was used as endogenous control for total and cytoplasmic proteins, and Lamin B1 was used as endogenous protein control for the nuclear proteins. (C) Apoptosis assays were performed in the various cancer cell lines transfected with NS or miR-99b-5p mimic in the absence or presence of 11nM docetaxel. Significantly different apoptosis capacity (* p-value < 0.05, in miR-99b-5p transfected cells treated with docetaxel vs. vehicle) were determined based on ANOVA with Tukey’s post-hoc test. Each value was represented as mean ± SD (n = 3–4); 231: MDA MB 231.