Background
Colorectal carcinoma (CRC) is the third most common cancer and one of the leading causes of death due to cancer world-wide [
1]. Defects in the apoptosis signaling cascade account for resistance to therapy of malignant tumors. In the case of CRC this resistance to radio- and chemotherapy substantially contributes to a poor prognosis. So far, the molecular mechanisms underlying the varying degree of cell death resistance of CRC are largely unknown. Therefore, a better understanding of the regulation of survival and therapy resistance of CRC cells is urgently required.
MicroRNAs (miRNAs) are short (20–23 nucleotides) non-coding mRNA molecules [
2] functioning as post-transcriptional regulators of gene expression. Since miRNAs contribute to the regulation of different cellular processes involving apoptosis, cell cycle regulation and differentiation, their deregulation quite often results in tumorigenesis [
3]. MiRNAs are transcribed as so-called pri-miRNAs, which are cleaved by Drosha in the nucleus [
4]. The resulting microRNA precursor molecules (pre-miRNAs) are subsequently transported into the cytoplasm and processed to the mature miRNA by the Dicer complex [
4]. The guide strand is integrated into the RISC complex resulting in the degradation of target mRNAs or their transcriptional inhibition [
4]. MiRNAs were first discovered in 1993 by Lee et al. [
5]. Since then, approx. 1400 human miRNAs have been discovered, amongst them almost 400 to be deregulated in CRC [
6].
So far, it has been shown that miR-210 is upregulated in a variety of human cancers, including lung cancer [
7‐
9], renal cell carcinoma [
10‐
13], pancreatic carcinoma [
14], breast cancer [
15], hepatocellular carcinoma [
16], colorectal carcinoma [
17,
18] and adrenocortical carcinoma [
19,
20]. Besides, miR-210 is downregulated in squamous cell carcinoma [
21] and ovarian cell carcinoma [
22]. Although miR-210 overexpression is accompanied by a poor prognosis in many human tumors [
15,
23‐
28], it has recently been shown, that high expression levels of miR-210 were significantly associated with an improved disease free survival in non-small cell lung cancer [
29] and clear cell renal cell carcinoma post nephrectomy [
30]. Similarly, low miR-210 expression levels were accompanied with a higher rate of relapse and a poorer treatment outcome in pediatric acute lymphoblastic leukemia [
31]. Further, the function of miR-210 regarding the regulation of cell growth and apoptosis is quite controversial. Whereas some studies show that downregulation of miR-210 reduces viability in renal cell carcinoma [
10], endothelial cells [
32] and hepatoma [
33], other studies claim that miR-210 acts in a pro-apoptotic manner in neuroblastoma [
34], lung adenocarcinoma [
35], renal cell carcinoma [
36], esophageal squamous carcinoma [
21] and lung adenocarcinoma [
37].
In this study, we sought to explore the functional role of miR-210 with regard to apoptosis in CRC. We demonstrate that an increased expression of miR-210 reduces proliferation, cell cycle progression and colony formation. Furthermore, overexpression of miR-210 induces ROS generation and apoptosis, accompanied by an increased Bim expression and Caspase 2 processing. Taken together our results identify miR-210 as a potent inducer of apoptotic cell death in CRC and suggest the miR-210-induced ROS generation to be a possible key player within this process.
Discussion
Altered expression of miR-210 can modulate either apoptosis resistance or sensitivity depending on the cellular context [
10,
21,
32‐
37]. MiR-210 has been shown to be upregulated in CRC compared to normal tissue [
17]. Further, high expression levels of miR-210 both in tumor tissue and serum of CRC patients correlate with a poor prognosis [
17,
18]. Within this study we sought to explore the functional effects of an increased miR-210 expression in CRC.
Our results indicate that miR-210 functions in an anti-tumorigenic manner by decreasing proliferation accompanied by an increased amount of cells in the G2/M phase of the cell cycle. This is in accordance with previous studies showing an accumulation of cells in the G2 phase in various tumor entities upon miR-210 overexpression [
35,
36,
48]. Moreover it has been shown that miR-210 overexpression induces senescence in fibroblasts [
49] and reduces tumor growth and proliferation in hepatocellular xenografts [
33]. Several direct and indirect targets of miR-210 might account for this effect. Zheng et al. previously demonstrated that overexpression of miR-210 blocks the expression of CyclinD1 and CyclinD2 via SHH/Gli1 signaling [
50]. Additionally, the miR-210 target E2F3 [
22,
36] plays an important role in regulation of proliferation [
51]. He et al. further proposed Plk1, CyclinF, Bub1B, CDC25B and Fam83D to be involved in miR-210-mediated cell cycle arrest [
48]. In addition to the direct targets of miR-210, proliferation arrest might be induced by an increased ROS generation. Several targets are known that are involved in cell cycle regulation and are regulated by ROS. The observed accumulation of cells in G2 phase might therefore be caused by the regulation of the oxidative state of Cdc25C. This phosphatase which activates cyclinB/cdk1 complexes, might be repressed by elevated ROS levels by inducing an inhibitory disulfide bond [
52]. Besides a direct regulation of cellular proliferation, elevated amounts of ROS might contribute to the observed effects via activation of FOXOs. These transcription factors regulate the transactivation of a series of genes involved in cell cycle control [
53]. Activation of the FOXO transcription factors might occur upon increased ROS levels by MST1, which is activated upon nuclear DNA damage [
54] or by inhibition of the AKT kinase, which negatively regulates FOXOs [
55]. Interestingly, we observed a decrease in phosphorylated AKT upon miR-210 overexpression. Similarly, Luo et al. recently demonstrated a ROS-dependent inactivation of AKT signaling accompanied with an increased activity of FOXO3a in colorectal cancer cells [
56].
An increased generation of ROS upon miR-210 upregulation has so far been observed in colorectal carcinoma [
42], in adipose-derived stem cells [
57] and fibroblasts [
49]. Furthermore, it has been shown that an elevated expression of miR-210 reduces oxygen consumption and upregulates glycolysis in various tumor entities [
39,
40,
42,
58]. Within this context, it has been observed, that the activity of mitochondrial complex I [
39,
40] and II [
37] is impaired, resulting in an increased formation of ROS [
54]. These effects have been proposed to be based on miR-210 mediated regulation of the Fe-S cluster scaffold protein ISCU [
39‐
42], SDHD [
37], a subunit of the succinate dehydrogenase complex, COX10 [
42], a subunit of cytochrome c oxidase, and NDUFA4 [
37], a subunit of the NADH dehydrogenase 1 alpha subcomplex. In line with these observations we could also detect a decreased expression of ISCU and NDUFA4 (data not shown) upon miR-210 overexpression. Whereas siRNA-mediated downregulation of ISCU increased ROS generation and induced apoptosis, siRNA-mediated downregulation of NDUFA4 did neither alter ROS generation nor apoptosis rates (data not shown). However, ectopic overexpression of ISCU did not counteract miR-210 mediated apoptosis and ROS generation. Therefore, it might by very likely, that ISCU is only one of several miR-210 targets regulating ROS generation. This is also evidenced by the effects of ISCU downregulation, since the reduced expression of ISCU did not completely reach the extent of miR-210 overexpression with regard to ROS generation and apoptosis induction.
Although there are several studies investigating the effect of a modulated miR-210 expression on apoptosis, the underlying molecular mechanisms are far from clear. Within this study we provide evidence, that an increased ROS generation induced by miR-210 overexpression contributes to the apoptotic phenotype. One of the most common pathways contributing to ROS-induced apoptosis is the ASK1 signaling cascade. Induction of ROS results in the oxidation of the inhibitory protein thioredoxin. Subsequently ASK1 and the downstream stress kinases JNK and p38 get activated, whereas the latter are able to induce cell death [
59].
Besides the ASK1/JNK/p38 signaling axis, the transcription factor FOXO3 might contribute to the apoptotic effects upon ROS generation by transactivation of its target genes Bim, Bcl-6 and Noxa [
54]. Within this context, FOXO3 might be activated by MST1 or inhibited by the AKT kinase [
54,
55]. Accordingly, it was recently demonstrated that ROS-dependent inactivation of the AKT signaling pathway was accompanied by an increase in Bim expression levels [
56]. Indeed, we detected an increase of Bim expression levels upon miR-210 overexpression, which was at least partially due to transcriptional regulation. Besides, ROS-induced ER stress and subsequent activation of the transcription factor CHOP might also contribute to the elevated Bim expression [
38,
60]. However, siRNA-mediated downregulation of Bim did not (HCT116) or only slightly (SW480 and SW707) diminish miR-210-mediated apoptosis, pointing to a different mechanism triggering miR-210-mediated apoptosis. Furthermore, inhibition of ROS generation using NAC did not alter Bim expression levels, rendering a ROS-dependent regulation of Bim rather unlikely.
Interestingly, we observed a miR-210 mediated upregulation of the anti-apoptotic Bcl-2 protein in HCT116 and SW480 cells. So far it has been reported, that Bcl-2 overexpression can either inhibit or increase ROS induced apoptosis [
61‐
63]. In this regard, we observed, that ectopic overexpression of Bcl-2 significantly reduced miR-210 mediated apoptosis (data not shown). Within this context it is tempting to speculate whether an increased expression of miR-210 sensitizes cancer cells to Bcl-2 inhibitors.
Within this study we could further demonstrate, that overexpression of miR-210 results in an increased processing of Caspase 2. Induction of ROS has been shown to induce activation of Caspase 2 [
43,
44] in a both p53-dependent and –independent manner [
64,
65] which might further result in apoptosis by Caspase 2-mediated cleavage of Bid or by directly inducing the release of Cyt c, AIF and SMAC from the mitochondria [
66]. However inhibition of ROS generation did not alter Caspase 2 processing nor did Caspase 2 downregulation inhibit miR-210 induced cell death (data not shown).
The functional consequences of an increased miR-210 expression in cancer patients are so far unknown. It has been shown, that elevated miR-210 expression levels might either be beneficial [
29‐
31] or be accompanied by a poor prognosis [
15,
17,
18,
23‐
27]. However, it is still unclear whether the latter is directly caused by increased expression levels of miR-210. Since hypoxia is one of the main factors contributing to a poor prognosis of cancer patients [
67], elevated miR-210 expression levels, which are mainly regulated by HIF transcription factors [
25,
68,
69], might only be a bystander effect instead of directly influencing patients’ outcome. Therefore, the regulation of apoptosis by miR-210 might be of great biological relevance in CRC and warrants further investigation.
Methods
Materials
N-acetylcysteine was obtained from Sigma-Aldrich (St. Louis, MO, USA, A9165). The antibodies were obtained as follows: anti-actin (Chemicon, Billerica, MA, USA, 1501); anti-AKT (Cell Signaling, Danvers, MA, USA, 9272); anti-Bad (Santa Cruz, Dallas, TX, USA, sc-7869); anti-Bax (Santa Cruz, sc-493); anti-Bcl-2 (Santa Cruz; sc-509); anti-Bcl-XL (Cell Signaling, 2764); anti-Bim (Cell Signaling, 2933); anti-Caspase 2 (Cell Signaling, 2224); anti-Caspase 3 (Imgenex, San Diego, CA, USA, IMG-144A); anti-cIAP1 (R&D Systems, Minneapolis, MN, USA, AF8181); anti-GAPDH (Santa Cruz, sc-32233); anti-ISCU (Santa Cruz; sc-373694); anti-Mcl-1 (Santa Cruz, sc-819); anti-pAKT (S472/473) (Cell Signaling, 4058); anti-Puma (Cell Signaling, 4976); anti-XIAP (Cell Signaling, 2045).
Cell culture
The human colorectal cancer cell lines HCT116, SW480 and SW707 as well as the human breast cancer cell line MCF-7 were purchased from the American Type Culture Collection (ATCC, USA), maintained in RPMI medium (Life Technologies, Darmstadt, Germany) supplemented with 10 % fetal calf serum (Sigma-Aldrich), 1 mM glutamine, 25 mM glucose and 1 % penicillin/streptomycin (Life Technologies) and cultured at 37 °C in a 5 % CO
2 atmosphere. Cell lines were regularly tested for the presence of contamination using multiplex cell contamination test [
70] and authenticated by SNP profiling [
71].
Proliferation and clonogenicity assay
For the assessment of proliferation, cells were seeded into 6-cm culture dishes and counted after 24, 48 and 72 h using the trypan blue exclusion assay. For clonogenicity assays, 500 cells were seeded into 6–well culture dishes and incubated for 7 days prior to crystal violet staining and colony counting.
FACS analysis
For analysis of cell cycle distribution and cell death, colorectal cancer cell lines were stained with propidium iodide (PI) as described previously [
72].
For measurement of reactive oxygen species (ROS), colorectal cancer cells were seeded in 6-cm plates and transfected as indicated. Cells were incubated with the fluorescent H2DCF-DA (2,7-dichlorodihydrofluorescein diacetate; 5 µM; Biozol, Eching, Germany) for 30 min at 37 °C.
Cells were subjected to flow cytometry analysis using a Becton–Dickinson FACScalibur cytometer and Cell Quest Software.
Transfections
Colorectal cancer cells were transiently transfected with siRNA using Lipofectamine 2000 (Life Technologies). Pre-miR-210 (miR precursor; PM10516) and co-pre-miR (control; AM17110) oligonucleotides were obtained from Life Technologies and used in a concentration of 50 nM. Bim siRNA #1 and #2 were obtained from Thermo Scientific (Waltham, MA, USA, #D-004383-18, #D-004383-17) and used in a concentration of 25 nM. ISCU siRNA was obtained from Life Technologies (#s23908) and used in a concentration of 5 nM. A non-specific siRNA served as a control (Thermo Scientific, #D-001810-01).
The pcDNA3-ISCU plasmid was generated by PCR from the clone pENTR221-ISCU, provided by the ORFeome Collaboration (Genomics and Proteomics Core Facility, DKFZ, Heidelberg, Germany) using the following forward (F) and reverse (R) primers containing BamHI and XhoI restrictions sites: 5′- ATGCATGCATGGATCCACCATGGCGGCGGCTGGGGCT -3′ (F) and 5′- ATGCATGCATCTCGAGCAAGAAAGCTGGGTCCAATTTC -3′ (R). The PCR products were digested with BamHI and XhoI and cloned into the correspondent sites of pcDNA3-Flag. For the generation of stable transfectants, complete medium containing Geneticin® (G418, Invitrogen) at a concentration of 1.5 mg/mL was used to select stably transfected cells.
Adenoviral transduction
Mcl-1-AdV was produced as described previously [
73]. The control AdV consists of the empty AdV5 backbone and was kindly provided by Stefan Herzig (DKFZ, Heidelberg, Germany). CRC cells were incubated with recombinant AdVs directly after seeding using a multiplicity of infection of 10 (HCT116) or 200 (SW480, SW707).
Immunoblot analysis
Cellular lysate generation and immunoblot analysis were performed as described previously [
72]. Densitometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA,
http://www.rsb.info.nih.gov/ij/).
Quantitative PCR analysis
Quantitative real-time PCR was performed as described previously [
73]. Following primer pairs were used: Bim: 5′-CAACACAAACCCCAAGTCCT-3′ (forward), 5′-TCTTGGGCGATCCATATCTC-3′ (reverse); 18S: 5′-CATGGCCGTTCTTAGTTGGT-3′ (forward), 5′ ATGCCAGAGTCTCGTTCGTT-3′ (reverse).
For measurement of miRNA expression, total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Hilden Germany). Mature miRNAs were reversely transcribed using TaqMan® MicroRNA reverse transcription kit (Thermo Scientific) and TaqMan® MicroRNA Arrays (Thermo Scientific). Quantitative PCR analysis was performed using the TaqMan® Universal PCR Master Mix (Thermo Scientific) and a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All steps were carried out according to the to the manufacturer’s protocols.
Statistical methods
Significant differences were identified using the unpaired 2-sided Student t test. Throughout, p values <0.05 were considered significant and are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.
Authors’ contributions
KET was involved in acquisition of data, in study conception and design, in analysis and interpretation of data, and in drafting the manuscript. AF participated in study conception and design, in analysis and interpretation of data. TS, JR, and SS were involved in acquisition of data. SMG was involved in analysis and interpretation of data. WR participated in study conception and design, in analysis and interpretation of data, and in drafting the manuscript. All authors read and approved the final manuscript.