Background
MICAL1 is a member of molecules Interacting with CasL (MICAL) family discovered in 2002 [
1]. In spite of its wide distribution in the nervous system [
2], MICAL1 has been found expressed in various human normal cells as well as cancer cell lines, including melanoma and HeLa cells [
3,
4]. Combined with the characteristic of anti-apoptosis, MICAL1 has been proven to be involved in cancer cell growth and survival regulation [
3,
4]. MICAL1 has four conserved domains: an N-terminal flavin adenine dinucleotide (FAD) binding domain, a calponin homology (CH) domain, a Lin11, Isl-1 and Mec-3 (LIM) domain and a C-terminal coiled-coil (CC) domain, where the FAD domain is responsible for the major portion of the MICAL1’s function [
5]. Studies have showed that FAD domain of MICAL1 contains flavin mono-oxygenase activity and has the ability to produce ROS [
3,
6]. It has been well documented that increased oxidative stress and ROS production is crucial for breast cancer development and maintenance of its malignant state [
7,
8]. Results from our studies have also shown that when breast cancer cells receive signals from their microenvironment, such as EGF, LPA and hypoxia, ROS level in cells may increase and functions as second messengers in intracellular signaling cascades which induce their migratory and invasive properties [
9‐
11]. However, whether MICAL1 could influence cell metastatic property by regulating ROS level in breast cancer cells remains to be determined.
It is now known that MICAL1 displays an auto-inhibitory mechanism to control its biomolecular function. Normally, the MICAL1 CC domain binds to its LIM domain to mediate the auto-inhibition. Removal of the CC domain from MICAL1 or affect the binding of CC domain to its LIM domain may cause the activation of its mono-oxygenase domain, leading to ROS production and F-actin assembly alteration. Actually, MICAL1 is a highly regulated protein, the auto-inhibition state could be relieved by the interaction occur within CC domain and other proteins such as RAB1 and Plexin under various cellular conditions [
12,
13]. RABs are the largest family of small GTPases and involved in the control of intracellular membrane trafficking and cell motility through interaction with specific effector molecules [
14,
15]. By a yeast two-hybrid assay, previous study has systematically screened the ability of MICAL1 binding for all the members of Rab family and found that GTP-bound RAB35 was one of the few members which interacted strongly with MICAL1 [
16].
Like all GTPases, RAB35 activity is under tight control, which is mediated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that catalyze GTP exchange and hydrolysis, respectively [
14]. Notably, RAB35 has ample opportunities to influence diverse cell signaling, resulting in functional promiscuity on tumor initiation and progression, and the activity of RAB35 in tumor is of tremendous research interest. It has been shown that RAB35 functions as a tumor suppressor and attenuated signaling downstream of Arf6 [
17]. However, Studies on
Drosophila cultured cells have led to the suggestion that RAB35 may promote the assembly of actin filaments during bristle development and increase filopodia formation [
18]. Similarly, there are also report that RAB35 is over-expressed in ovarian cancer [
19]. Recent studies including the results from our laboratory also showed that RAB35 activation could be act as a positive regulator of cell shape, phagocytosis as well as migration in various types of cells [
20‐
22]. Several studies have highlighted a link between RAB35 and MICAL-l1, a similar protein to MICAL1, which revealed that RAB35 could use MICAL-l1 as its membrane hub effector [
23,
24]. Although RAB35 could recruit different effectors to perform specific biological process, it remains unclear whether and if so, the biological relevance of RAB35 binding to MICAL1 in breast cancer cells. In this study, we examined whether knockdown or overexpression of MICAL1 could influence ROS generation and cell migration firstly, and then explored the mechanism underlying MICAL1 action by examining the effect of RAB35 blockage/activation on those process.
Methods
Cell and plasmids
Human breast cancer cell lines MDA-MB-231, MCF-7, T47D, BT474 and MDA-MB-468 were obtained from the Cell Biology Institute of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) (Hyclone, Thermo Scientific, Waltham, MA, USA) supplemented with 10 % (v/v) fetal bovine serum (FBS) (Hyclone) and antibiotics (100 U/mL streptomycin and 100 μg/mL penicillin) (Invitrogen, Carlsbad, USA) in a humidified incubator at 37 °C with 5 % CO2. Cells were grown on coverslips for fluorescence staining and on plastic dishes for protein extraction. Cells were made quiescent by serum starvation overnight followed by EGF (R&D Systems, Minneapolis, MN, USA) treatment.
The RAB35-Q67L (constitutively active, CA), RAB35-S22N (dominant negative, DN) and wild-type RAB35 (WT) plasmids were kindly provided by Dr. Matthew P. Scott (Department of Developmental Biology, Stanford University, USA). The PCR products were cloned into the pEGFP-N1 vector (Clontech, Palo Alto, CA, USA). Human MICAL1 cDNA clone was purchased from Youbio (Hunan, China). The full-length MICAL1 DNA was amplified from pOTB7-MICAL1 plasmid using the following primer set, sense: 5′-CCC
AAGCTTGCCACCATGGCTTCACCTACCTCCA-3′, antisence: 5′-CCAA
CTCGAGGCCCTGGGCCCCTGTCCCCAAGGCCA-3′. In these primers, Hind III and Xho I restriction site sequences have been underlined. The polymerase chain reaction (PCR) products were cloned into the pCMV-C-HA vector (Beyotime, Nantong, China). Truncated MICAL1 lacking CC domain (residues 1–799) and truncated MICAL1 containing CC domain (residues 800-1068) were also created as previously described [
3]. The cells were seeded in 6-well plates, cultured to 80 ~ 90 % confluence, and then transiently transfected with those plasmids by using FuGENE HD Transfection Reagent (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions.
siRNA knockdown studies
The sequences of small interfering RNA (siRNA) for MICAL1 were as follows: #1, 5′-GUCUCUGCCUUUGACUUCATT-3′, #2, 5′-CUGCAGAACAUUGUGUACUTT-3′, and #3, 5′-CUCGGUGCUAAGAAGUUCUTT-3′; siRNA for RAB35 was: 5′-GCAGCAACAACAGAACGAUTT-3′ and the sequence of control siRNA was 5′-UUCUCCGAACGUGUCACGUTT-3′ (GenePharma, Shanghai, China). Cells were transfected with siRNA by Lipofectamine 2000 according to the manufacturer’s instruction.
Migration and invasion assays
For wound healing assay, breast cancer cells were seeded in a 96-well plate. Approximately 24 h later, when cells were 95 ~ 100 % confluent, cells were incubated overnight in DMEM and wounding was performed by scraping through the cell monolayer with a 10 μl pipette tip. Medium and nonadherent cells were removed, and cells were washed twice with PBS, and new medium with or without EGF was added. Cells were permitted to migrate into the area of clearing for 18 h. Wound closure was monitored by visual examination under microscope (Carl Zeiss Meditec).
For transwell migration assay, breast cancer cells in exponential growth were harvested, washed, and suspended in DMEM without FBS. Cells (2 × 105/200 μl) were seeded into polycarbonate membrane inserts (8 μm pore size) in 24-transwell cell culture dishes. Cells were allowed to attach to the membrane for 30 min. The lower chamber was filled with 600 μl DMEM with EGF or with 10 % FBS. Cells were permitted to migrate for 12 h. After the incubation, stationary cells were removed from the upper surface of the membranes. The cells that had migrated to the lower surface were fixed and stained with 0.1 % crystal violet. Cell invasion was analyzed using the same protocol as for cell transwell migration, but with the use of matrigel (BD Bioscience) pre-coated cell culture inserts. Cells were permitted to invade for 24 h.
Coimmunoprecipitation and immunoblotting assays
Coimmunoprecipitation assays were performed as previously described. Briefly, cell lysates were incubated with antibody at 4 °C overnight. Antibody-bound complexes were precipitated with protein A + G agarose beads (Beyotime) and eluted by rinsing buffer, then the agarose-associated protein complexes were dissolved in SDS loading buffer and analyzed by immunoblotting assays.
Sample protein extraction and concentration determination of whole cells were performed as previously described [
9]. Briefly, equal amounts of protein were run on SDS polyacrylamide gels and transferred to nitrocellulose membrane. The resulting blots were blocked with 5 % non-fat dry milk and probed with antibodies. The following antibodies were used: GAPDH (KangChen), MICAL1 (proteintech) (Santa Cruz Biotechnology), RAB35 (BD Biosciences) (ABclonal Technology), Akt, P-Akt, HA and GFP antibodies (Cell Signaling). Protein bands were detected by incubating with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Millipore).
Pulldown assays
RAB35 activity was measured by pulldown assays as described previously [
22]. In brief, the GST fusion RBD35 was purified from BL21 bacteria and incubated with cell lysates. Then the complexs were incubated with MagneGST Glutathione Particles (Promega) for 30 min on a rotating wheel at 4 °C. After washed with washing buffer and collected by magnet in a magnetic stand (Promega), the beads were solubilized in 2 × SDS loading buffer, and then subjected to immunoblotting assays with antibody against RAB35.
Immunofluorescence and immunohistochemistry assays
Cells used for immunostaining were fixed in ice-cold methanol for 20 min, permeabilized in 0.1 % Triton X-100 and blocked in PBS containing 1 % BSA for 1 h at room temperature. The cells were incubated with primary antibody overnight at 4 °C followed by incubation with FITC or rhodamine conjugated secondary antibody for 1 h at room temperature within a moist chamber. After wash with PBS, the samples were mounted with DAPI Fluoromount G (Southern Biotech). Images were acquired using an Olympus BX51 microscope coupled with an Olympus DP70 digital camera.
Measurement of ROS
For intracellular H2O2 staining, 1 × 105 cells were seeded on a coverslip placed in a 6-well plate and incubated overnight. After treated with appropriate inhibitors and stimuli as detailed elsewhere in the text, the cells were stained with 5 μM 2′,7′-dichlorofluorescein diacetate (CM2-DCFHDA) (Invitrogen) for 15 min at 37 °C. After wash with PBS, the cover slips were mounted on glass slides. Images were collected using an Olympus BX51 microscope coupled with an Olympus DP70 digital camera.
The level of superoxide anions in the cells was measured using the enhanced lucigenin chemiluminescence method. The homogenate supernatant of total cellular protein was diluted in modified HEPES buffer. The reaction started by addition of 5 μM dark-adapted lucigenin (Sigma). Light emission was measured for 10 times in 10 min with a luminometer (20/20n, Turner), and average values were calculated and expressed as mean light unit (MLU) per min per milligram of protein, which represented the level of superoxide anions.
Statistical analysis
Statistical analysis was performed using the SPSS statistical software program (Version 19.0; SPSS, Chicago, IL, USA). Error bars represent standard error of mean S.E.M, the significance of difference in two groups was analyzed by Student’s t test. P < 0.05 represents statistical significance and P < 0.01 represents sufficiently statistical significance (two tailed).
Discussion
While there is only one gene encoding MICAL in
Drosophila, vertebrates contain three genes encoding MICAL isoforms indicated as MICAL1, MICAL2 and MICAL3. Furthermore, MICAL-like forms, which were absent of FAD domain, also have been identified exist in vertebrates. In
Drosophila, MICAL selectively oxidizes Met 44 residue within the D-loop of actin, thereby destabilizing F-actin and inhibiting local assembly [
25]. MICAL1 has the most closely related domain architecture to
Drosophila MICAL [
3], however, to date, only a few reports have been published to describe the functions of MICAL1 during cancer progression. Previous studies have shown that aberrant activation MICAL1 is a negative regulator of apoptosis and contributes to malignant progression of melanoma [
4]. In the present study, we demonstrate that knockdown of MICAL1 has favorable effect on preventing cell migration and invasion. We also show that ROS acts as downstream of MICAL1 to regulate cell invasion. Moreover, we determine a novel link between RAB35 and MICAL1 in regulating EGF-induced breast cancer cell invasion. Taken together, we demonstrate for the first time that MICAL1 may play a potential role in breast cancer cell motility and shed light on new therapeutic target against breast cancer invasion and metastasis.
Enhanced motility of cancer cells is a critical ability in promoting tumor metastasis and mortality of patients. Here, we delineated the role of MICAL1 in regulating breast cancer cell motility. Our results showed that the migratory and invasive ability of breast cancer cells induced by EGF or FBS stimulation decreased significantly after MICAL1 silencing in vitro. Consistently, MICAL1 overexpression in the cancer cells accelerated their motility behavior. Recent study showed that MICAL2-positive cells peculiarly localized at the primary human gastric cancer invasive front and MICAL2 knock-down in cancer cells resulted in mesenchymal to epithelial transition [
26]. Similar with those results, in the present study, we uncovered an essential role of MICAL1 in promoting migration and invasion of breast cancer cells. Given our observation that silencing MICAL1 specifically inhibited EGF induced cell invasion, it will be interesting to elucidate the exact mechanisms by which EGF regulate MICAL1’s function.
ROS are highly reactive molecules generated by incomplete reduction of oxygen, including superoxide, hydrogen peroxide, and hydroxyl radical et al. Of note, increased oxidative stress and ROS production was present in many human metastatic tumors, and the roles of ROS in triggering signaling pathways for cell migration and invasion have been well established [
27,
28]. Here, we demonstrated that the increased level of ROS production after EGF stimulation was markedly blocked by silencing of MICAL1. Moreover, cells only expressing CC domain from MICAL1 displayed lower levels of ROS when compared with cells overexpressed full-length of MICAL1. Our finding is consistent with a previous report that HeLa cells transfected with the FAD domain from MICAL1 augmented ROS levels [
3]. Consistently, the levels of ROS were significantly attenuated upon transfection of the enzymatically impaired FAD domain mutant [
3]. Therefore, it is proposed that during EGF stimulation, MICAL1, especially for its FAD domain, facilitates the production of ROS, helping to promote the migratory and invasive ability of breast cancer cells.
As due to their very nature, ROS cannot impart cell migratory functions directly. Activation of PI3K/Akt by ROS was shown be an important mechanism to mediate breast cancer cell migration by LPA [
9]. In keeping with this idea, MICAL1-induced breast cancer cell invasion might be PI3K/Akt dependent. Our observations have yielded evidence that the increase of P-Akt was markedly blocked after the silencing of MICAL1. Consistently, P-Akt was higher in MICAL1 overexpressed breast cancer cells. It is worth noting that the effect of ROS on lung cancer cell migratory functions is dependent on Akt activity [
29]. Here, we also found that P-Akt level as well as cell invasion was blocked by application of ROS scavenger NAC. Therefore, Akt is more likely the target of ROS downstream of MICAL1 to regulate breast cancer cell motility.
RAB35 may functions downstream of growth factor receptors and associates with PI3K. Further, the expression of GTP-bound RAB35 is necessary and sufficient for PI3K/Akt signaling activation and apoptosis resistance in human tumors [
30]. Our previous work suggested a link between RAB35 activity and increased breast cancer cell migration [
22]. Although some studies showed that RAB35 has the opposite effect on the migration in some kind of cancer cells [
17,
31], in the present work, we found that EGF induced RAB35 activation, while blocking RAB35 expression greatly abolished EGF-induced cell invasion. These results suggest that EGF might promote cell invasion in breast cancer cells by activating RAB35. Until now, limited knowledge was concerning the regulation of MICAL1 function by EGF signaling in breast cancer cells. In the current study, we determined that RAB35 and MICAL1 coimmunoprecipitated, and this interaction was disrupted when RAB35 was inactivated. We also observed that MICAL1 silencing delayed the increased invasive ability of RAB35 (CA)-expressing breast cancer cells. Moreover, knockdown RAB35 reduced ROS level as well as P-Akt level in breast cancer cells. Besides the fact that CC domain of MICAL-l1 interacts with active mutants of RAB35 [
23], and the inhibitory effect of MICAL on ROS generation is thought to be dependent on the binding of CC domain to its LIM domain, therefore, we speculated that active form of RAB35 might be able to release MICAL1 auto-inhibition by directly binding to the CC domain of MICAL1, thereby allowing ROS generation and promoting cell migratory and invasive properties.
Abbreviations
CC, Coiled-coil; CH, Calponin homology; EGF, Epidermal growth factor; FAD, Flavin adenine dinucleotide; FBS, Fetal bovine serum; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GAPs, GTPase activating proteins; GEFs, Guanine nucleotide exchange factors; LIM, Lin11, Isl-1 and Mec-3; MICAL1, Molecules interacting with CasL; qRT-PCR, Quantitative real time polymerase chain reaction; ROS, Reactive oxygen species; SDS-PAGE, Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Acknowledgements
Not applicable.