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Apoptosis

, Volume 23, Issue 7–8, pp 388–395 | Cite as

Treating metastatic prostate cancer with microRNA-145

  • Alexandre Iscaife
  • Sabrina Thalita Reis
  • Denis Reis Morais
  • Nayara Izabel Viana
  • Iran Amorim da Silva
  • Ruan Pimenta
  • Andre Bordini
  • Nelson Dip
  • Miguel Srougi
  • Katia Ramos Moreira Leite
Article

Abstract

Prostate cancer (PCa) is an incurable disease at the metastatic stage. Although there are different options for treatment, the results are limited. MicroRNAs (miRNAs) are a group of small, noncoding, regulatory RNAs with important roles in regulating gene expression. miR-145 is reported to be a key tumor suppressor miRNA (tsmiR) that controls important oncogenes, such as MYC and RAS. In this study, in vitro studies were performed to show the control of MYC and RAS by miR-145. Flow cytometry was used to analyze cell proliferation and apoptosis. The efficacy of miR-145 in treating metastatic PCa was tested in nude mice using a model of bone metastasis promoted by intraventricular injection of PC-3MLuc-C6 cells. Tumor growth was evaluated by an in vivo bioluminescence system. After the full establishment of metastases on day 21, six animals were treated with three intravenous doses of miR-145 (on days 21, 24 and 27), and six were injected with scramble miRNA as controls. Compared to the controls, tumor growth was significantly reduced in animals receiving miR-145, most importantly on day 7 after the third and last dose of miRNA. After discontinuing the treatment, tumor growth resumed, becoming similar to the group of non-treated animals. A decrease in MYC and RAS expression was observed in all cell lines after treatment with miR-145, although statistical significance was achieved only in experiments with LNCaP and PC3 cell lines, with a decrease in 56% (p = 0.012) and 31% (p = 0.013) of RAS expression, respectively. Our results suggest that miR-145 is a potential molecule to be tested for treatment of metastatic, castration-resistant PCa.

Keywords

Prostate cancer Metastasis Therapy MicroRNA Molecular biology 

Introduction

Nearly 14% of men are diagnosed with prostate cancer (PCa) during their lifetime, and 3% will die of the disease. Although the 5-year overall survival of PCa is approximately 100%, the rate drops to 28% if PCa progresses to the metastatic stage [1].

The treatment for metastatic PCa is based on blocking androgen signaling, and this approach is typically effective for an average of 3 years. After that time, several mechanisms promote tumor growth, although serum testosterone levels remain low. Although new drugs have shown good results, such as Bicalutamide [2] and Enzalutamide [3], this phase of PCa, referred to as castration-resistant disease, still presents great challenges to oncologists and urologists, so the development of new and effective treatments is urgent.

MicroRNAs (miRNAs) are a group of small noncoding regulatory RNAs, ~ 22 nucleotides in length, that play a fundamental role in the regulation of gene expression [4]. They are involved in the regulation of multiple cellular processes, including proliferation, differentiation, cellular stress responses and apoptosis. miRNAs can act as tumor suppressor genes (tsmiRs) or oncogenes (oncomiRs), depending on their targets, and changes in the biogenesis and expression of miRNAs are associated with tumor development and the progression of several types of neoplasia, including prostate [5, 6].

There are reports evaluating the use of miRNAs for the treatment of tumors exploring different miRNAs and technologies for drug delivery; some are already in clinical or preclinical phases of development [7].

For PCa, there are few studies reporting reduction in tumor growth, sensitization of tumors to cytotoxic drugs or immunotherapy by local or systemic injection of miRNAs in animal models [8].

In 2008, Mercatelli et al. demonstrated that the subcutaneous injection of anti-miR-221/222 in a localized prostate cancer animal model reduced tumor growth [9]. Takeshita et al. evaluated a model of prostate cancer with bone metastases using an in vivo image detection apparatus to analyze systemic treatment with miR-16, and the study demonstrated promising results [10, 11].

Downregulation of miR-145 has been repeatedly described as a tsmiR in many tumors, including prostate, inhibiting critical oncogenes such as MYC, RAS, IRS-1 and ERG [12, 13, 14, 15, 16, 17, 18, 19].

Our aim was to analyze miR-145 as a potential therapeutic agent to treat metastatic PCa in an animal model after confirming its negative control over important oncogenes by reducing MYC and RAS expression and ultimately influencing apoptosis and cell cycle.

Materials and methods

In vitro studies

The metastatic prostate cancer cell lines, DU145 (brain metastasis) and PC3 (bone metastasis), were obtained from ATCC, and LNCaP cells (lymph node metastasis) were kindly provided by UNICAMP (State University of Campinas) and were maintained in DMEM, RPMI and MEM medium, respectively.

The cell lines were transfected with miR-145, anti-miR-145 or their respective negative controls (scramble miRNA) using the transfection agent, Lipofectamine (Thermo Fisher Scientific, MA USA).

The cell transfections of Pre-miR™ and Anti-miR™ were conducted according to the Pre-miR™ miRNA Precursor Protocol (Ambion, Austin, TX, USA). We analyzed the expression of the KRAS (Hs00364284_g1) and cMYC (Hs00153408_m1) genes using qRT-PCR after transfection.

To confirm the efficiency of miRNA transfection, the expression level of miR-145 was detected by qRT-PCR (Hs002278).

For apoptosis detection and cell cycle analysis, flow cytometry (Attune Flow Cytometer) was used after treatment with Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen™—556547) or propidium iodide (PI), respectively.

All experiments were performed in triplicate for statistical analysis.

In vivo study: tumor induction

This study was approved by the Ethics and Animal Research Committee of our institution under protocol number 0053/13.

The cell line PC-3M-luc-C6, PC-3M-luc-C6 is a luciferase-expressing cell line that is derived from PC3M human adenocarcinoma cells by stable transfection of the North American firefly luciferase gene expressed from the SV40 promoter and was acquired from Caliper Life Sciences (Hopkinton, MA, USA) and cultured in MEM medium supplemented with glutamine.

Male athymic nude (Balb/c) mice, 9–11 weeks of age, were anesthetized with 50–70 µl of ketamine and lidocaine. Tumor cells were administered via a left ventricular intra-cardiac injection of 100 µl of a Dulbecco’s PBS sterile solution containing 2 × 106 PC-3M-luc-C6 cells using a 1 cc syringe and a 30G/6 mm needle (BD®). The puncture was performed in the 3rd/4th left intercostal space, 3 mm lateral to the sternum and 15 degrees from the horizontal plane with the needle directed towards the animal’s left shoulder.

To check the efficiency of the dissemination of tumor cells, d-luciferin (Promega) (5 mg/kg) was injected into the peritoneum immediately after the intra-cardiac injection, and the dissemination of the cells was confirmed by the IVIS equipment (Xenogen - IVIS® Spectrum). The animals were used for further analysis if D-luciferin injections revealed that more than 90% of the body was bioluminescent. The animals were then examined weekly by the IVIS system, with day 0 (D0) designated the day of the intra-ventricular injection. Metastases were detected at approximately week 3 (D21), and animals presenting diffuse metastases were subjected to the experimental treatments.

miR-145 treatment

Atelocollagen (Koken®, Tokyo, Japan) was used to stabilize the miRNA as previously described [7]. The treatment solution comprised equal volumes of miR-145 and atelocollagen (0.1% in PBS, pH 7.4) at a final concentration of 0.05% [13]. On days 21, 24 and 27 following the injection of the tumor cells, 200 µl solution containing 3 mg/kg of miRNA plus atelocollagen was injected into the tail veins of the animals. Six animals were injected with miR-145, and six were injected with the scramble miRNA negative control.

The animals were examined weekly by IVIS and the photons were counted as previously described [10]. On day 48, the animals were euthanized, and their organs were analyzed microscopically.

Statistical analysis

Statistical analysis was performed using SPSS 19.0 software (IBM Corp. Armonk, NY). Differences in gene expression between transfected cells and controls were analyzed using the formula, \({{\text{2}}^ - }^{{\Delta \Delta {C_{\text{T}}}}}\). Proliferation and apoptosis were analyzed by Student´s t test for parametric data and the Mann–Whitney U test for non-parametric data. The photon emission detected by IVIS equipment, corresponding to the size of tumors each day, was analyzed by Student´s t test comparing treated and controls after the one-sample Kolmogorov–Smirnov test showed homogeneous distribution of the results. The differences in growth were based on the mean of photon emission on day 0 (baseline—21st day after tumor cell injection), day 1 (first day of treatment), day 3 (3rd dose of treatment), 7 and 14 days after the interruption of treatment. For all statistical analyses, we considered a level of significance of 5% (p < 0.05).

Results

In vitro studies

The efficiency of miR-145 transfection was evaluated by qRT-PCR comparing the miR-145 transfected DU145 cells with those transfected with the scramble control. The levels of miR-145 in transfected cells was variable from 56.66 to 224.93, mean 154.30.

The evaluation of apoptosis after miR-145 transfection showed an increase in apoptosis by 24% in DU145 cells (p = 0.008) compared with the control, and no change in apoptosis was observed in LNCaP or PC3 cells treated with miR-145. In PC3 cells, miR-145 inhibited proliferation by 18% compared with the control (p = 0.01). A marginal significant difference was observed in gene expression assays after treatment with miR-145 and anti-miR-145 for DU145 and LNCaP cells.

The treatment with miR-145 showed a 48% reduction in the expression of cMYC, while DU145 cells treated with anti-miR-145 had an increase of 82% in cMYC expression (p = 0.08). In the LNCaP cells, miR-145 treatment reduced cMYC expression by 29%, whereas anti-miR-145 treatment increased cMYC expression by 16% (p = 0.056). In PC3 cells, miR-145 reduced cMYC expression by 33%, but no difference was detected in the control (p = 0.136). The expression of KRAS decreased by 48% in DU145 cells treated with miR-145 and increased by 83% in DU145 cells treated with anti-miR-145 (p = 0.105). In LNCaP cells, KRAS expression decreased by 56% following miR-145 treatment and increased by 12% in LNCaP cells treated with anti-miR-145 (p = 0.012). KRAS expression decreased by 31% in PC3 cells treated with miR-145 and increased by 18% when treated with anti-miR-145 (p = 0.013) (Fig. 1).

Fig. 1

Expression of cMYC and KRAS detected by qRT-PCR after transfection of miR-145 and anti-miR-145 in PC3, DU145 and LNCaP cell lines

In vivo studies

Animals injected with tumor cells developed multiple metastases, primarily in the mandible, ribs, femur and iliac bones. Metastases to solid organs were also identified in the lungs, adrenal glands and in the liver.

Treatment with miR-145 decreased the rate of tumor growth, as observed in the bioluminescence emission curves (Figs. 2, 3, 4). The reduction in the growth rate was first observed after the initial dose, and maximal growth inhibition was observed on day 34, 7 days after the discontinuation of treatment. The mean emission was almost 3 times higher in the control group, 8, E + 07 in treated animals versus 3, E + 07 in control animals (p = 0.036). However, the inhibitory effect on growth was transient, and after day 34, tumor growth resumed at the same rate as the control group. This comparison revealed that metastases in the control animals grew substantially and continuously and that the size and location of the lesions in the miR-145-treated animals remained relatively stable (Fig. 5).

Fig. 2

Mean bioluminescence detected by IVIS in six animals treated with miR-145

Fig. 3

Mean bioluminescence detected by IVIS in six animals treated with scramble miRNA as controls

Fig. 4

Curves show the mean bioluminescence of all mice from group treatment and controls. The lesion growth of the control group is constant and higher, compared to the treated animals. On the seventh day after interruption of treatment, the difference was significantly different, being smaller in the treated group

Fig. 5

Images obtained by IVIS equipment in two animals representative of treated and controls. The bioluminescence was obtained from each lesion and for comparison, we used the mean emission of all lesions, first in each animal and at the end, in all animals from each group

Weight loss was observed in both groups, although greater weight loss was observed in the control group. There was a mean loss of 20% of initial body weight in the control group versus 16% in animals treated with miR-145.

In both groups, the necropsy examination revealed diffuse metastases in the bones, and less frequently in the liver, lungs and adrenal glands. Although the evaluation of toxicity was not the subject of this study, the microscopic evaluation of lungs, liver and kidneys did not show any signs of toxicity.

Discussion

This is the first study to show therapeutic efficacy of miR-145 in a metastatic PCa animal model. We showed a decrease in tumor growth during miR-145 treatment. On the seventh day after the interruption of treatment, which consisted of three doses of miR-145, there was a significant reduction in the size of metastasis compared to the control group. As expected, tumor growth resumed when the treatment was discontinued. This pattern was similar to that observed with other target drugs, such as those related to tyrosine kinase signaling and the mTOR pathway [20].

In previous studies, we evaluated the expression of miRNAs associated with various stages of prostate cancer progression, from high-grade prostate intraepithelial neoplasia to metastatic disease. We observed a loss of expression of multiple miRNAs during tumor progression, including miR-145 [12, 21]. Similar results were observed using a microarray technique and genome-wide association analysis (GWA) in radical prostatectomy specimens. miR-145 was downregulated in patients who had experienced biochemical relapse after undergoing a prostatectomy [22].

These findings prompted us to further evaluate miR-145 for in vitro and in vivo studies. The in vitro studies confirmed that miR-145 acts as a tumor suppressor miRNA (tsmiRNA) by inhibiting the expression of the cMYC and KRAS oncogenes in three prostate cancer cell lines, inducing apoptosis in DU145 cells and inhibiting proliferation in PC3 cells.

The expression of miR-145, located on chromosome 5, is induced by p53 primarily in response to cellular stress. In most tumors, p53 is a key tumor suppressor related to metastatic progression. miR-145 targets several genes, including MYC, RAS, IRS-1, and ERG and is downregulated in PCa tissues and in PCa cell lines [12, 23].

Treatment with miR-145 showed reduction in tumor growth, although a drastic effect was not achieved. Some aspects related with the design of the study can be responsible for this result. A previous study published by Takeshita et al. [11] evaluated the efficacy of miR-16 in a metastatic PCa model. In their study, tumor injection occurred (day 0), and miRNA treatment was then initiated via the tail vein on days 4, 7 and 10 before metastatic disease detection. One potential drawback of this experiment was that it was not clear whether metastasis was successfully induced. In our model, treatment was initiated after metastases had developed, ensuring that any changes in tumor behavior were due to the miRNA treatment.

Although the effect of miR-145 was limited, it was similar to what has been observed with other targeted therapies for advanced cancer, namely, that tumor growth was inhibited during treatment, but the growth resumed after treatment discontinuation [24, 25, 26, 27]. Even though our model might be perceived as a faithful reproduction of human metastatic disease, an alternative approach to evaluate miR-145 efficacy to treat metastatic PCa would be to begin treatment immediately after metastases are first observed.

No standards for the appropriate dose or duration of treatment have been reported due to a lack of studies, and no pharmacodynamic or pharmacokinetic data have been reported to date. We used a dose of 3 mg/kg of highly purified miRNA in this study, which was the same used in the in vivo experiments reported by Takeshita [11]. We believe that higher doses and prolonged treatment might inhibit tumor growth for a longer period of time and might even promote disease regression. The animals in this study were administered 3 doses of treatment at intervals of 3 days. Prolonged treatment might have continued to suppress tumor growth, but the fragility of the tail vein precludes the evaluation of the treatment over a prolonged period of time.

The histological examination of organs by microscopy did not reveal any hepatic, renal or lung lesions, which we inferred as an absence of treatment-associated toxicity. In future studies, biochemical exams should be performed to evaluate the safety parameters associated with systemic injections.

In conclusion, for the first time, we showed therapeutic activity of the tumor suppressor miR-145 in treating metastatic PCa. In the future, this miRNA could be used as monotherapy or as a component of combination therapy to treat metastatic prostate cancer.

Notes

Acknowledgements

We thank Mara Sa Junqueira for support with the animals. We thank Cristina Massocco Sales Gomes and Rodrigo Tannura for technical support with cell cycle assays. We thank Hernandes Faustino Carvalho for important reagents. We thank Jose Pontes Junior and Marcia Kubrusly for the critical reading of this manuscript. This study was supported by São Paulo Research Foundation (FAPESP) grants #2012/21966-8 and #2013/07350-7.

Author contributions

AI: Designed and performed experiments, analyzed data, performed statistical analysis and participated in writing the manuscript. STR: Designed experiments and participated in writing the manuscript. DRM: Performed experiments. NIV: Performed experiments. IAS: Performed experiments. AB: Participated in writing the manuscript. ND: Designed experiments. MS: Designed experiments, analyzed data and participated in writing the manuscript. KRML: Designed experiments, analyzed data and participated in writing the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Alexandre Iscaife
    • 1
  • Sabrina Thalita Reis
    • 1
  • Denis Reis Morais
    • 1
  • Nayara Izabel Viana
    • 1
  • Iran Amorim da Silva
    • 1
  • Ruan Pimenta
    • 1
  • Andre Bordini
    • 1
  • Nelson Dip
    • 1
  • Miguel Srougi
    • 1
  • Katia Ramos Moreira Leite
    • 1
  1. 1.Laboratorio de Investigação Medica da Disciplina de Urologia – LIM 55, Faculdade de Medicina FMUSPUniversidade de Sao PauloSão PauloBrazil

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