miR-30e* is overexpressed in prostate cancer and promotes NF-κB-mediated proliferation and tumor growth

Abstract

According to the CDC prostate cancer (CaP) has the highest incidence and second highest mortality rate amongst cancers in American men. Constitutive NF-κB activation is a hallmark of CaP and this pathway drives many pro-tumorigenic characteristics of CaP cells, including cell proliferation and survival. An activated NF-κB gene signature is predictive of CaP progression and biochemical recurrence following therapeutic intervention. However, the mechanisms that perpetuate NF-κB activation are incompletely understood. Genes that control NF-κB activity are rarely mutated in CaP suggesting that epigenetic mechanisms may contribute to constitutive NF-κB activation. microRNAs (miRs) epigenetically regulate many genes involved with NF-κB activation. IκBα is a direct inhibitor of NF-κB; it binds to and sequesters NF-κB in the cytoplasm resulting in functional inhibition. IκBα is a target gene of miR-30e* yet the expression and oncological impact of miR-30e* in CaP is unknown. We report that miR-30e* expression is elevated in multiple murine models of CaP and is most pronounced in late stage disease. miR-30e* drives CaP proliferation and tumor growth through inhibition of IκBα, which results in chronic activation of NF-κB. Additionally, we show that inhibition of miR-30e* improves chemotherapeutic control of CaP. Thus, miR-30e* may prove to be a novel clinical target whose inhibition leads to decreased CaP cell proliferation and sensitization of CaP cells to chemotherapeutics.

Keywords: NF-κB; cyclin D1; microRNA; prostate cancer.

Figure 1. miR-30e* expression is elevated in CaP

(A) Whole prostates were harvested from TRAMP mice at 6-, 8-, 12 and 29-weeks of age and corresponding age matched control C57BL/6J mice (n = 3). (B) Prostates were also harvested from Hi-MYC mice along with wild type FVB age matched control mice (n = 2). Prostates were analyzed for miR-30e* and U6 snRNA expression via qRT-PCR. Raw data was analyzed and displayed in graph using the 2^−dCq formula. Welch's t-test (A) and Student t-tests were performed (B), Error bars represent SEM; * P ≤ 0.05, ** P ≤ 0.01.

Figure 2. miR-30e* regulates CaP cell proliferation

(A) C2H cells or (B) PC3M cells were transfected with either miR-30e* inhibitor oligos (■) or control scramble oligos. Twenty-four and forty-eight hours later MTT assays were performed. Results are reported as % viability relative to viability observed in cells transfected with control scramble oligos; each time point of the experiments was repeated a minimum of 4 times. Welch's t-tests were performed, Error bars represent SEM;* P ≤ 0.05, ** P ≤0.01, *** P ≤ 0.001, **** P ≤0.0001. (C) Cell senescence was tested by staining either control or miR-30e* inhibited C2H cells for β-galactosidase, hydrogen peroxide treated fibroblasts were used as a positive control (Positive Control). Positively stained cells were analyzed in three separate 200x fields of view; counts were repeated 3 times and the average of the counts was recorded. Results are reported as # of senescent cells / field. Welch's t-tests were performed, error bars represent SEM; n = 3, P > 0.05. (D) Cell apoptosis was tested by detecting cleaved caspase-3 via ELISA in control or miR-30e* inhibited C2H cells, TSA treated JAR cells were used as a positive control (Positive Control Cells). Results are reported as pg/mL of cleaved caspase 3. Welch's t-tests were performed, error bars represent SEM; n = 3, P > 0.05. (E) Cell proliferation was tested by IHC staining of Ki67 in control or miR-30e* inhibited C2H cells. Positively stained cells were analyzed using a light microscope in three separate 200x fields of view; counts were repeated 3 times and the average of the counts is presented as % of Ki67 positive cells. Welch's t-tests were performed, error bars represent SEM; n = 3, * P ≤ 0.05.

Figure 3. miR-30e* regulates NF-κB activity which is essential for prostate cancer cell viability

(A) To evaluate NF-κB activation, an NF-κB luciferase reporter construct was transfected into C2H cells. Luciferase was then evaluated in control and miR-30e* inhibited cells 24 hours post inhibition. Results are depicted as NF-κB activity relative to control experimental group. Welch's t-tests were performed, error bars represent SEM; n = 4, * P ≤ 0.05. To assess whether NF-κB regulates prostate cancer viability TRAMP C2H (B) and PC3M (C) cell viability was assessed following a 24 hour treatment with 10 μM, 50 μM and 100 μM Bay 11-7085 treatment via MTT analysis. Equal volume ethanol carrier was used as a control. Welch's t-tests were performed, error bars represent SEM, n = 3, *P ≤ 0.05 and **P ≤ 0.01.

Figure 4. miR-30e* regulates prostate cancer cell proliferation and tumor growth through IκBα

(A) Plasmid maps for doxycycline inducible N-terminal HA tagged miR-30e* sensitive IκBα and miR-30e* resistant IκBα pTetOne plasmids. (B) To evaluate whether the miR-30e*: IκBα interaction regulates CaP cell proliferation, C2H cells were transfected with a doxycycline inducible vector that expresses either miR-30e* sensitive or resistant IκBα. Cell viability was assessed in both miR-30e* sensitive (●) and resistant (■) clones following stimulation with 100 ng/mL doxycycline hyclate via MTT assay. Results are depicted as fold change relative cells not treated with doxycycline. Welch's t-tests were performed, error bars represent SEM; n ≥ 3,* P ≤ 0.05, ** P ≤0.01. To determine if the specific targeting of IκBα by miR-30e* regulated NF-κB p65 activation miR30e* resistant IκBα C2H cells were administered subcutaneously in C57BL/6 mice (C). Once the tumors reached 100mm3, mice were randomized and initiated on either dox chow or remained on control chow. Tumor were explanted once the tumor volume reached 800mm3 and nuclear NF-κB p65 was quantified via IHC. Five sections were quantified from each tumor (n=4, P ≤ 0.001). Normal (●) or doxycycline containing (■) chow tumors were monitored and tumor growth was assessed. Results are depicted as tumor growth (mm³) after doxycycline treatment (D) and percent survival (E). Welch's t-tests were performed, error bars represent SEM; n ≥ 6, *** P ≤ 0.001.

Figure 5. The miR-30e*: IκBα axis regulates cyclin D1 and proliferation in vivo

The expression of cyclin D1 (A) and Rb (total and phosphorylated) (B) were evaluated in C2H cells treated with or without miR-30e* inhibitor for 24h via western blot analysis. Representative blots are shown on the right; summary results are depicted as expression relative to control C2H cells. Welch's t-tests were performed, error bars represent SEM; n ≥ 4, * P ≤ 0.05. To assess if miR-30e* regulates prostate cancer cell proliferation and the expression of cyclin D1 in vivo IHC was performed. miR30e* resistant IκBα C2H cells were administered subcutaneously in C57BL/6 mice. When the tumors had established and reached 100mm³, normal (●) or doxycycline containing (■) chow was administered, tumors were harvested, formalin fixed and stained for Ki-67 (C) and cyclin D1 (D) when they reached 800 mm³. Images (C-D) represent IHC staining from 2x representative tumors from both normal and doxyxline chow fed mice and are displayed at 20x. (E) C2H cells were treated with either docetaxel alone (●) or in combination with miR-30e* inhibitor (■) and viability was assessed via MTT assays. Results are depicted as % viable (absorbance value of experimental relative to untreated TRAMP C2H cells). Welch's t-tests were performed, error bars represent SEM; n = 4, * P ≤ 0.05, ** P ≤ 0.01.

Figure 6. The microRNA-30e*: NF-κB axis regulates prostate cancer cell proliferation and therapeutic resistance

miR-30e* targets IκBα mRNA thus increasing NF-κB activation. NF-κB drives chemotherapeutic resistance and also increases cyclin D1 production. Cyclin D1:CDK4/6 phosphorylates pRb allowing it to dissociate from E2F. Free E2F pushes CaP cells from G1 to S phase and drives proliferation and therapeutic resistance.