Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1

It has been shown that the presence of MLH1 seems to be not only most important for PMS2 stabilization [30] but also for other interacting partner proteins [26]. Since we previously identified protein-protein interaction of MLH1 and SPTAN1 [12] and verified this interaction using HEK293 cells (Additional file 1), we analyzed the influence of MLH1 on the protein level of interacting SPTAN1 by using nine different human cancer cell lines: six MLH1 proficient and three MLH1 deficient ones. The MLH1 proficient cell lines were the human epitheloid cervix carcinoma cell line HeLa, the human embryonic kidney cell line HEK293, two stably MLH1 transfected clones of the colorectal carcinoma cell line HCT116, HCT116 mlh1-2 and HCT116 mlh1-3 [31] as well as the colorectal carcinoma cell lines SW480 and LoVo. We compared the SPTAN1 expression level of these cell lines with their corresponding MLH1 deficient cell lines, HEK293T [32] or the stably pcDNA3.1+ mock control transfected HCT116 mlh0-1 cell line [31], respectively and with the MLH1 deficient colorectal cancer cell line RKO using Western blot analysis.

As shown in Figure 1, MLH1 deficient cell lines (Figure 1, lanes 2, 5 and 7) showed significantly reduced SPTAN1 expression in comparison to the MLH1 proficient lines (Figure 1, lanes 3, 6, 8 and 9).

Most prominent SPTAN1 expression level was detectable in MLH1 proficient HeLa cells (Figure 1A, lane 1) which was used as the benchmark for the quantification of SPTAN1 expression of all tested cell lines and set to 100% (Figure 1B, lane 1).

SPTAN1 expression of HCT116 mlh0-1 (Figure 1, lane 2) was only 49% compared to HeLa and 31% less than the SPTAN1 level of the HCT116 mlh1-2 clone (Figure 1, lane 3). Even weakly MLH1 expressing HCT116 mlh1-3 cells (Figure 1, lane 4) showed 21% stronger expression of SPTAN1 than the MLH1 deficient mock control (Figure 1, lane 2).

In MLH1 deficient HEK293T cells (Figure 1, lane 5) SPTAN1 expression was only 44% in comparison to HeLa and 34% lower than the MLH1 proficient HEK293 (Figure 1, lane 6). Moreover, MLH1 deficient RKO cells (Figure 1, lane 7) expressed only 44% of SPTAN1 in comparison to HeLa and significant less than the MLH1 proficient SW480 and LoVo cells (Figure 1, lane 8 and 9).

In order to analyze the influence of MLH1 knock down on the mRNA level of SPTAN1 we determined the mRNA amount of SPTAN1 48 h after treatment in comparison to control siRNA treated cells via qRT-PCR (Figure 2C and D).

We forced MLH1 protein reduction via siRNA treatment with two different siRNAs against MLH1 using the MLH1 proficient cell lines of our panel: HeLa, HCT116 mlh1-2, HCT116 mlh1-3, HEK293, SW480 and LoVo (described above). As demonstrated by Western blot analysis (Figure 2A and B) siRNA knock down of MLH1 lead to SPTAN1 reduction after 48 h in all these cell lines (Figure 2A and B, lanes 3, 6, 9, 12, 15 and 18) whereas treatment with siRNA control had no effect on SPTAN1 expression (Figure 2A, lanes 2, 5, 8, 11, 14 and 17).

However, siRNA knock down of MLH1 led not only to the reduction of MLH1 itself but also to reduced SPTAN1 mRNA levels in four or five of the treated cell lines, respectively (Figure 2C and D, lanes 3, 6, 9, 12 and 18).

In addition, the MLH1-SPTAN1 connection was verified by monitoring SPTAN1 expression in parallel to growing amount of MLH1. Hereby, SPTAN1 was determined in HEK293T cells transiently transfected with MLH1 from 0 h to 20 h. As shown in Additional file 2, a clear increase of SPTAN1 expression was detectable (Additional file 2A and C, lanes 6-9) slightly time-delayed to the gain of MLH1 (Additional file 2A and B, lanes 4-9).

To emphasize that MLH1 itself and not a general loss of the MMR system lead to SPTAN1 reduction, we also determined SPTAN1 levels in MMR deficient LoVo cells caused by a defect in MSH2 (the second most important Lynch syndrome associated MMR protein [33]). However, similar protein levels of SPTAN1 were detectable in MSH2 deficient as well as MSH2 overexpressing LoVo cells (Additional file 3), therefore an influence of MSH2 on SPTAN1 could be excluded.

Since deregulation of SPTAN1 was described to cause changed cellular SPTAN1 localization in sporadic CRCs [21], we furthermore compared the cellular distribution of SPTAN1 in nuclear and cytoplasmic fractions of MLH1 proficient SPTAN1 transfected and untransfected HEK293 as well as MLH1 deficient HEK293T cells (Figure 3). Our data show that SPTAN1 was expressed in both cell fractions of all tested cell lines (Figure 3, middle panel) and overexpression of SPTAN1 generally led to enhancement of SPTAN1 similarly in the cytoplasm as well as in the nucleus. However, while the amount of SPTAN1 was equal in the cytoplasm and the nucleus of both MLH1 proficient HEK293 variants, MLH1 deficient HEK293T showed obviously less SPTAN1 in the cytoplasm than in the nuclear fraction (Figure 3, lane 3 vs lane 6).

To verify the connection of MLH1 and SPTAN1 in vivo, we isolated protein extract of one MLH1 deficient fresh CRC biopsy and the corresponding normal tissue from a patient. Additionally, we performed immunohistochemistry using paraffin embedded invasive growing CRC tissue from patients showing defective MLH1 expression in comparison to those with MLH1 proficiency. Hereby, we analyzed sporadic MLH1 deficient CRCs, CRCs from Lynch syndrome patients caused by MLH1 mutations and sporadic CRCs without MLH1 defect (Figure 4).

In the fresh biopsy tissue from the CRC patient, SPTAN1 was well detectable in the normal tissue (Figure 4E, lane 2) while the corresponding tumor showed reduced expression (Figure 4E, lane 1).

Immunhistochemistry data (Table 1) demonstrated a significant reduction of SPTAN1 in all MLH1 deficient tumor sections, while SPTAN1 could be clearly detected in all MLH1 proficient CRC tissues; exemplary results are shown in Figure 4B and D, respectively.

To test the potential functional influence of SPTAN1 reduction on cell mobility the migratory rate of MLH1 deficient and MLH1 proficient cells was compared in a migration assay by measuring the alteration of the wound width at different time points (3 h, 6 h, 12 h and 15 h). As shown in Figure 5A and B, mobility of HCT116 mlh0-1, HCT116 mlh1-2, SPTAN1 overexpressing HCT116 mlh0-1 as well as siRNA SPTAN1 treated HCT116 mlh1-2 cells grown in 96-well plates was analyzed. The mobility of MLH1 deficient HCT116 mlh0-1 cells was significantly worse than that of the MLH1 proficient HCT116 mlh1-2 sister clone (Figure 5A and B). Overexpression of SPTAN1 enhanced the migration ability of MLH1 deficient HCT116 mlh0-1 to a similar extent as detected for the MLH1 proficient sister cell line (Figure 5A and B). siRNA knock down of SPTAN1 in HCT116 mlh1-2 cells leads to a similar mobility as detected in MLH1 deficient HCT116 mlh0-1 cells (Figure 5A and B). Treatment of HCT116mlh1-2 with control siRNA showed no effect (data not shown).

This effect could also be shown by using doxycycline inducible HEK293T cells (Figure 5C and D). Doxycycline treated MLH1 deficient HEK293T cells showed worse mobility that untreated MLH1 proficient HEK293T cells (Figure 5C and D).

Paraffin-embedded tissue from 11 patients with surgically resected well characterized CRC specimens were selected for immunhistochemical analysis: 4 were from Lynch syndrome patients caused by pathogenic MLH1 mutations, 4 were sporadic CRCs with MLH1 defect caused by hypermethylation of the MLH1 promoter associated with BRAF V600E mutation and 3 tumors were MMR proficient sporadic CRCs.

Characteristics of the analyzed patients-tissue are summarized in Table 1.

MLH1 as well as SPTAN1 expression was analyzed in triplicate, respectively, of every tumor and tumor surrounding tissue using immunohistochemistry.

In parallel, a fresh (MLH1 promotor hypermethylated) tumor biopsy and the corresponding normal tissue were exemplarily taken from one CRC patient to perform Western blot analysis of MLH1 and SPTAN1.

The study was approved by the local ethic committees of Frankfurt/Main (Germany), Bonn (Germany) and Homburg Saar (Germany). All patients gave informed consent.

HeLa (ATCC number CCL-2), HEK293 (ATCC number: CRL-1573) and LoVo (ATCC number: CCL-229) were purchased from American Type Culture. HEK293T cells, obtained from Dr. Kurt Ballmer (Paul Scherer Institute, Villingen, Switzerland) were grown in DMEM with 10% FCS. As previously described, MLH1 is not expressed in HEK293T [32]. Stably transfected HCT116 cells: HCT116 mlh 0-1 transfected with the pcDNA3.1+ vector and HCT116 mlh 1-2 as well as HCT116 mlh 1-3 transfected with pcDNA3.1+ containing the entire open reading frame of MLH1 obtained for Prof. Francoise Praz (Institute Gustave Roussy, Villejuif, France) were grown in DMEM with 10% FCS and hygromycin B (100 μg/ml) [31]. Doxycycline inducible HEK293T MLH1+ cells, obtained from Prof. Josef Jiricny (University of Zurich, Institute of Molecular Cancer Research, Zurich, Switzerland), were cultured in DMEM supplemented with 10% Tet System-approved fetal calf serum, hygromycin B (300 mg/ml), and Zeocin (100 mg/ml). To abrogate MLH1 expression, the cells (HEK293T MLH1-) were cultured for 4 days in the presence of doxycyclin (50 ng/ml) [43].

Anti-SPTAN1 (C-11), anti-Lamin B (C-20) and anti-MSH2 (H-300) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), anti-MLH1 (G168-728) as well as anti-gamma Adaptin (88) were from Pharmingen (BD Biosciences, United States), anti-beta Actin (Clone AC-15) was purchased from Sigma (Sigma-Aldrich, USA) and anti-SPTAN1 (MAB1622) was from Millipore (Millipore, USA).

The pcDNA3.1+/MLH1, pcDNA3.1+/PMS2 and pcDNA3.1+/MSH2 expression plasmids were described previously [27]. Full length SPTAN1 cDNA was generated from total RNA of human lymphocytes and subcloned into the eukaryotic expression vector pcDNA3.1- via the KpnI/XhoI restriction sites.

All plasmids were confirmed by sequencing and reading frames were corrected using site-directed mutagenesis, if necessary. All oligonucleotides were purchased from Sigma-Aldrich (Munich, Germany).

Different stably transfected HCT116 cells (HCT116 mlh 1-2 and HCT116 mlh 1-3), HEK293 and HeLa cells were treated with siRNA according to manufacturer’s protocol (Applied Biosystems, Foster City, CA). In brief, 100 μl of OPTI-MEM (Gibco, Biocompare, San Francisco, CA) was mixed with 5 μl siPORT NeoFx Transfection Agent (Applied Biosystems, Foster City, CA) and incubated for 10 min at room temperature. 100 μl of OPTI-MEM containing 30 nM of Silencer Validated siRNA MLH1 (siRNA1) (Applied Biosystems, ID# 119549), 5 nM of Silencer Select siRNA MLH1 (siRNA2) (Ambion, ID# 4392420), 5 nM of Silencer siRNA SPTAN1 (Applied Biosystems, ID# s13405), or 30 nM of Silencer Negative Control siRNA (Applied Biosystems, ID#1), respectively was added and incubated for further 10 min at room temperature. 1×10⁵ cells, diluted in DMEM medium with 10% FCS, were added to a final volume of 2.5 ml, the mixture was placed in a 6-well and incubated at 37°C with 5% CO₂ for 24 h and 48 h. Thereafter, treated HCT116 mlh 1-2 and HCT116 mlh 1-3, HEK293 and HeLa cells were harvested, homogenized in Trizol reagent (Invitrogen, Carlsbad, CA) and RNA was isolated according to manufacturer’s protocol. The mRNA levels of MLH1, SPTAN1, or GAPDH (internal control) were determined by a real-time quantitative reverse transcription-PCR (qRT-PCR) assay (TaqMan) with a pair of MLH1-, SPTAN1- or GAPDH-specific primers and MLH1-, SPTAN1- or GAPDH-specific probes (#Hs00179866-m1 MLH1, #Hs00162203_m1 SPTAN1, #Hs02786624_g1 GAPDH, Applied Biosystems, Foster City, CA), respectively, on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA).

Transiently transfection or cotransfection of HEK293T cells was carried out as described previously [26]. In brief, HEK293T were transfected at 50-70% confluence with expression plasmids, pcDNA3.1+/MLH1, pcDNA3.1+/PMS2, pcDNA3.1-/SPTAN1 or empty pcDNA3.1+ mock control (1 μg/ml, respectively) using 10 μl/ml of the cationic polymer polyethylenimine (Polysciences, Warrington, PA; stock solution 1 mg/ml). 24 h and 48 h post transfection cells extracts were prepared for Western blot analysis.

Separation of proteins from cell cultures into nuclear and cytoplasmic fractions was carried out as previously described [35]. Briefly, cells were harvested by centrifugation (10 min, 1000 g, 4°C), cell pellets were washed twice in PBS (4°C) and diluted in 250 μl hypotonic buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl_2, 0.5 mM DTT). After incubation on ice (15 min), 5% of NP-40 (10%) was added, samples were vortexed and centrifuged (10 min, 1000 g, 4°C). Supernatants containing cytoplasmic proteins were frozen and residual pellets were resuspended in cell extraction buffer (10 mM Tris/HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% Na-depoxycholate, 1 mM PMSF and 5% protease inhibitor cocktail (Sigma Aldrich, Munich, Germany)). Samples were incubated on ice (30 min), sonicated (10 sec) and centrifuged (13000 U/min, 4°C, 30 min). Dissolved nuclear protein fractions were frozen (-80°C).

Fresh tissue biopsy sections (à 5 mg) were removed from 80°C, washed three times with PBS (4°C), transferred into a 15 ml Dounce Homogenizer tube and diluted with 150 μl (4°C) ready to use Cell Lysis Reagent (Sigma Aldrich, Munich, Germany). Biopsies were homogenized vigorously (10 min) on ice, transferred into 1.5 ml Eppendorf tubes, sonicated (10 sec) and centrifuged (5 min, 1000 g, 4°C). Supernatants containing the proteins were placed in new Eppendorf tubes and frozen (-80°C).

Proteins were separated on 10% polyacrylamide gels, followed by Western blotting on nitrocellulose membranes and antibody detection using standard procedures or as described previously [26].

The band intensity of SPTAN1 was quantified using Multi Gauge V3.2 program (Fujifilm, Tokyo, Japan).

All experiments were performed in quadruplicate.

Coimmunoprecipitation was carried out as previously described [12]. For detection of endogenous protein-protein interaction coimmunoprecipitation was carried out using anti-MLH1 and anti-SPTAN1, respectively, in MLH1 proficient HEK293 cells. Immunoprecipitation with protein A/G sepharose served as negative and whole cell extract (50 μg) of HEK293 cells as positive control.

MLH1 and SPTAN1 expression was analyzed by immunohistochemistry using paraffin embedded invasive growing MLH1 deficient or MLH1 proficient colorectal tumor tissue. Immunohistochemical analysis of MLH1 expression was carried out as described before [44].

For SPTAN1 detection, sections (2 μm) of representative samples were cut from paraffin embedded invasive growing colorectal carcinoma specimens. Normal colon mucosa served as an internal control. Sections were deparaffinized three times with Xylene and rehydrated in graded alcohol baths. Antigen retrieval by heating was required in a pressure cooker for 2 min with EDTA buffer, pH 8.0. This was followed by incubation of 5 min with 3% H₂O₂ for blocking endogenous peroxidase. Before and between different incubation steps, the sections were washed with 1× Envision Flex Wash Buffer (Dako, Germany). Primary antibody (Santa Cruz; mouse monoclonal antibody clone C-11; dilution 1:250) were diluted in Antibody diluent (Zytomed). Sections were incubated with the primary antibody for 30 min at room temperature followed by application of the EnVision-system (DakoCytomation) with horseradish peroxidase as enzyme and 3,3′diaminobenzidine tetrahydrochloride as chromogen. The sections were counterstained with Mayers hematoxylin (Applichem).

Immunhistochemical staining was examined using a Keyence microscope (Model BZ 9000 for a magnification of 100×, KEYENCE Co., Osaka, Japan).

Prior to initiating the migration assay, 1×10⁴ cells per well were seeded in 96-well Essen ImageLock plates (Essen Bioscience, Ann Arbor, Michigan, USA) and grown for 48 h to confluence under standard conditions. Then, cell-free zones were generated by using a 96-pin WoundMaker (Essen Bioscience, Ann Arbor, Ml) to simultaneously create wounds in all wells.

Thereafter, the plates were placed inside an automated microscope (IncuCyte™ (Essen Instruments)) which resides inside a standard cell culture incubator and equilibrated for 2 h before the first scan.

The cells were scanned every 3 h and the width of the cell-free zone was determined by IncuCyte™ software which is capable to identify the exact wound region.

To calculate the exact distance of cell migration the detected wound width of each time point was subtracted from the first measured wound width in the corresponding well. This value was divided by 2 and indicates the length (μm) of migration.

In parallel pictures were taken by IncuCyte from each well at every time point. The experiment was performed in triplicate.