MASTL induces Colon Cancer progression and Chemoresistance by promoting Wnt/β-catenin signaling

The human colon cancer cell lines HCT116, SW620, SW480, HT29, DLD-1, CaCo2, Ls174T, and IEC-6 cells were obtained from ATCC (Manassas, VA, USA) and cultured in RPMI-1640 containing 10% fetal bovine serum and 1% antibiotic and antimycotic (thermoFisher). Cells were transfected as described previously using effectene reagent [4]. Mastl-knockdown cell population was selected using puromycin (1 mg/ml). The activated β-catenin (S33Y) mutant was described previously [5].

RNA from human samples was hybridized to Affymetrix Human Genome U133 Plus 2.0 GeneChip Expression Array.The protocols and procedures for the procurement of human tissue samples and details of the microarray platforms and statistical analysis have been described previously [6, 7].

These analyses were performed using the standard protocols as described before [4]. Anti-MASTL Antibody (clone 4F9, MABT372, EMD Millipore), anti-E-cadherin antibody (BD transduction laboratories, USA), β-catenin (BD transduction laboratories, USA), GSK3beta (Cell Signaling Technology, Danvers, MA,USA), p-GSK3beta (Cell Signaling Technology, Danvers, MA,USA) Bcl-xL (Cell Signaling Technology), Survivin (Cell Signaling Technology) and anti-b- actin (Sigma, St. Louis, MO), were used for immunoblotting.

To assess cell proliferation, MTT assay was performed as described previously [7]. Anchorage-independence growth assay were used to determine the growth potential of MASTL knockdown cells as described previously [7].

Oncogenic array analysis was performed using proteome profiler human xl oncology array kit (R&D Systems, Minneapolis, MN)) as per manufacturer’s instructions.

Invasive potential of cells was measured in transwell filter insert with 8.0 μm pore polycarbonate membrane (Corning) coated with Matrigel (BD, Franklin Lakes, NJ, USA) as described previously [7].

The 5-ethynyl-2′-deoxyuridine (EdU), a thymidine analogue, is incorporated into cellular DNA during DNA replication [8]. The incorporated EdU can be detected through a reaction between ethynyl group of EdU and a fluorescent azide in a copper-catalyzed [3 + 2] cycloaddition (“Click” reaction) using Click-iT™ EdU imaging kit (Invitrogen, Carlsbad, CA) as per manufacturer protocol.

CaspACE™ Assay System (Promega Corp., Madison, WI) was used to detect caspase-3 activity as per manufacturer protocol.

We used the Hoechst/annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) triple staining detection system to assess cell apoptosis. FITC Annexin V Apoptosis Detection Kit II (BD Biosciences, San Jose, CA) was used as per the manufacturer’s instructions.

Total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN) according to manufacturer instructions as described [7].

Transfected cells were harvested and plated in six-well plates and cultured for 72 h in serum-free medium after which cells were treated with RO3306 (Sigma, St. Louis, MO), a CDK1 inhibitor for 16 h. After 16 h, media was replaced with fresh media and cells were grown for 1 h, and then fixed and cell cycle analysis was carried out. The percentage of cells in G0/G1, S, and G2/M phases of the cell cycle was determined using flow cytometer (FACS Calibur, BD Biosciences, San Jose, CA) after PI staining.

All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of UNMC. The tumorigenicity of cells under study was assessed using subcutaneous flank inoculation of 1 × 10⁶ cells in 6-week-old athymic nude mice. Animals were assessed for 5 weeks after the inoculation for tumor incidence and growth and then were sacrificed Tumor volume was measured using the formula Tumor volume = 1/2(length × width²)/2 as previously described [2, 7].

To characterize the potential role of MASTL in colon carcinogenesis, we first assessed its expression in a high through-put transcriptome analysis of a large patient cohort (combined Moffitt Cancer Center/Vanderbilt Medical Center expression array data set using 250 CRC patient tumors and 10 normal adjacent tissue samples as described previously [demographics; [7]]). We found robust stage-specific up-regulation of MASTL transcript levels compared to normal adjacent mucosal specimens (Fig. 1a; P < 0.001). We found a similar significant increase in MASTL expression in all stages of colon cancer compared to normal samples, analyzing the TCGA database (Fig. 1b).

To examine if the observed increase in MASTL mRNA expression was translationally relevant, we determined MASTL expression in a panel of colorectal cancer cell lines, animal models of colon cancer as well as a commercial colon cancer tissue array [immunohistochemical (IHC) analysis; 50 samples]. We found that immunoblotting using lysates from confluence matched cell lines indeed demonstrated a similar increase in MASTL expression in colon cancer cells versus non- transformed intestinal epithelial cells (IEC-6) (Additional file 1: Figure S1A). IHC analysis also revealed marked increase in MASTL expression in CRC tissue samples (compared to normal colon; Fig. 1c). To corroborate these findings, we further determined MASTL expression in colon tumor samples from a murine model of sporadic colon cancer (CRC; the APCmin mice) and colitis- associated cancer (CAC; AOM/DSS induced mouse model). A significant increase in MASTL expression was observed in tumors from both colon cancer models (Fig. 1d). These findings validated a positive association between MASTL expression and colon cancer progression.

We further determined whether high MASTL expression could also identify high-risk colon cancer patients. Overall survival estimates based on MASTL expression were determined in the CRC patient database as described previously [7]. We used a median cut-off for MASTL expression (higher-than-median = high MASTL expression; lower-than-median = low MASTL expression). We noted a significant association of better overall survival for patients with lower-than-median MASTL expression (Fig. 1e, p = 0.03) while patients exhibiting high MASTL expression had worse overall survival. We found a similar trend using disease-specific survival as an outcome measure (Fig. 1f, p = 0.05). Our additional analysis, wherein we divided patients into four quartiles based on MASTL expression values and performed Kaplan-Meier analysis, revealed a similar trend (Additional file 1: Figure S1B). These data rendered strong evidence that MASTL can serve as a prognostic biomarker for latent disease aggressiveness among colorectal cancer patients.

In further studies, to identify the causal significance of MASTL expression in CRC progression and the specific tumorigenic trait that is affected by MASTL expression, we evaluated tumorigenic and invasive properties of HCT116 and SW620 cells in response to inhibition of MASTL expression. Selection of cell lines for these studies was based on known tumorigenic/metastatic potential and high MASTL expression. Anti-human MASTL shRNA was expressed in these cells and silencing efficiency was confirmed by qRT-PCR, immunoblotting and immunofluorescence analyses (Fig. 2a (i&ii), Additional file 1: Figure S2). HCT116MKD and SW620MKD cells (with inhibition of MASTL expression) were analyzed using the anchorage-independent growth and matrigel-coated transwell-based invasion assays. Inhibition of MASTL expression significantly inhibited cell invasion (P < 0.05) and the ability of these cells to form colonies in soft agar by 60–80% (P < 0.05) {Fig. 2b (i&ii) and Additional file 1: Figure S3}. These data supported a necessary role of MASTL in promoting the oncogenic and metastatic properties of colon cancer cells.

To ascertain specific cellular function affected by MASTL expression for these observed changes, we performed cell cycle analysis using HCT116MKD and SW620MKD and respective control cells.

Confluent cell monolayer was serum-starved for 72 h to achieve cell synchronization.

Thereafter, cells were treated with RO3306 (10 μM), widely used to arrest cell cycle at G2/M interphase [8] for 16 h. Cells were released into the cell cycle by exposing them to fresh medium for 1 h. At this time, cells were collected for cell cycle analysis. As shown in Fig. 3, control cells progressed in the cell cycle to the G0/G1 phase. MASTL knockdown cells, however were unable to overcome the G2/M block by RO3306 so as to enter mitosis. As expected, beyond this, the percentage of cells in G2/M was 3–5 fold higher in HCT116MKD cells (Fig. 3a, p < 0.05) and SW620MKD cells (Fig. 3b, p < 0.05) compared to respective control cells.

G2/M arrest can lead to apoptosis in various cancer cell lines, including colon cancer cells [9‐11]. We thus next determined whether MASTL knockdown modulated proliferation or apoptosis by inhibiting the cell cycle at the G2/M interphase. To determine potential changes in cell- proliferation, MASTL knockdown and control cells were subjected to Edu-incorporation assay where it was observed that inhibiting MASTL expression significantly inhibited proliferative capacity of HCT116MKD and SW620MKD cells (Fig. 4a (i&ii)). To determine potential changes in apoptosis, cells were subjected to annexin-V and caspase activity assays. The annexin-V analysis revealed significant increases in both early and late apoptosis in HCT116MKD (Fig. 4b(i)) and SW620MKD cells (Fig. 4c(i)) compared to control cells (p < 0.05). Caspase 3/7 activity was also increased − 3-4 fold in these cells, suggesting a necessary role for MASTL in the regulation of processes determining cell multiplication and death (Fig. 4b (ii), C(ii)).

To further examine the precise molecular mechanisms modified by genetic silencing of MASTL expression to induce aggressive phenotype, we performed an analysis of the global changes in protein expression, especially for proteins implicated in promoting oncogenesis, using a well-controlled and commercially available oncogenic array (Additional file 1: Figure S3). We focused on proteins which were reproducibly altered in both colon cancer cells (HCT116^MKD and SW620^MKD). As shown in Additional file 1: Figure S4, we found consistent and significant alterations in the expression of Survivin and Bcl-xL, key proteins of anti-apoptotic pathways and upregulated in cancer cells [12‐15], in MASTL-silenced cells versus control cells. We further confirmed significant downregulation of Bcl-xL and Survivin expressions using immunoblotting in MASTL-inhibited colon cancer cells compared to respective controls (Fig. 5a, b). Of note, Survivin is one of the downstream target genes of the Wnt/β-catenin signaling pathway [15, 16]. A critical significance of hyper-activated Wnt/β-catenin signaling in colon tumorigenesis is well established [16, 17]. Moreover, β-catenin expression is elevated during the G2/M interphase of the cell-cycle progression [18, 19]. We therefore reasoned that there might be a causal correlation between MASTL and Wnt/β-catenin signaling in promoting colon carcinogenesis through modulating Survivin and/or Bcl-xL expressions. Therefore, we further determined the effects of MASTL-knockdown upon β-catenin expression, cellular localization, and transcriptional activity (Fig. 5c (i&ii)). We found that knockdown of MASTL also resulted in sharp decreases in β-catenin expression, as well as expression of c-Myc, a Wnt/β-catenin signaling target gene. Further determinations demonstrated marked decreases in the nuclear accumulation of β- catenin and transcriptional activity, as measured by the TOP-Flash reporter activity, in MASTL inhibited colon cancer cells. In contrast, forced overexpression of full-length MASTL cDNA in colon cancer cells induced sharp increases in the expression of these proteins (Additional file 1: Figure S5). To see if similar correlation between these proteins existed in CRC patient samples, we interrogated expression levels of the c-Myc, Bcl-xL and MASTL in the 260-patient CRC database used to determine MASTL expression in CRC. It was quite encouraging that levels of c-Myc and Bcl-xL (BCL2L1) expression associated with MASTL in a similar fashion in patient samples as noted in vitro (Additional file 1: Figure S6).

Of importance, glycogen synthase kinase 3 (GSK3), in complex with Axin and adenomatous polyposis coli (APC), phosphorylates β-catenin at Thr41, Ser37, and Ser33. Phosphorylated β-catenin is specifically recognized by β-TrCP, a subunit of the SCF^β-TrCP E3 ubiquitin ligase complex. The SCF^β-TrCP ubiquitin ligase poly-ubiquitinates β-catenin, leading to β-catenin degradation via the proteosome pathway [20].

In contrast, phosphorylated and/or inactive GSK-3β promotes cellular accumulation and nuclear translocation of β-catenin, and initiation of the T-cell factor (Tcf)-dependent transcription. In agreement with this, GSK-3β phosphorylation (S9, inactive form) was seen to be markedly reduced in MASTL knockdown cells, resulting in active GSK-3β to induce β-catenin degradation (Fig. 5d). Further analysis showed no significant change in β-catenin mRNA expression, well in accordance with potential post-transcriptional regulation (data not shown).

To determine if inhibiting MASTL expression can similarly modulate colon tumorigenesis in vivo, we performed a subcutaneous xenograft tumor assay using HCT116MKD and respective control cells in athymic nude mice (n = 6/group). The same mice received control and MASTL knockdown cells on opposite flanks. In line with previous reports [7], mice receiving HCT116C cells demonstrated tumor development as early as two weeks post-injection of cancer cells, and average tumor volume was 1068 ± 161.2 mm3 at 4-weeks post-injection. By contrast, tumors resulting from injection of HCT116MKD cells were significantly smaller, with average volumes of 309 ± 50.6 mm3 after the same period of growth (Fig. 6a, c). Tumor weight followed a similar pattern and was lower (P < 0.05) in mice injected with MASTL-inhibited cells compared to those injected with control cells (Fig. 6b). Resulting tumors were then evaluated for expression of MASTL, β-catenin, Survivin, and Bcl-xL expression. Also effects of MASTL inhibition on cell proliferation, and apoptosis in tumors were determined (Fig. 6d, e). Similar to in vitro findings, MASTL inhibition reduced expression of β-catenin, Survivin and Bcl-xL expression in tumors resulting from HCT116MKD cells. Further, an increased rate of apoptosis, as determined by cleaved PARP expression, while decreased proliferation as determined by Ki67 immunoreactivity in tumors resulting from HCT116MKD was observed. This suggests that inhibiting MASTL expression restores a cell death program and inhibits proliferation. These data from xenograft tumor assays provide further support for the role of MASTL in tumorigenesis in CRC.

Encouraged by these findings, we asked whether effects on apoptosis mediated by MASTL can be ameliorated by simply upregulation of β-catenin expression. We overexpressed a mutant β-catenin (S33Y) construct (which resists proteosomal degradation and thus is highly stable) in both HCT116MKD and SW620MKD cells. Overexpression of β-catenin and its expected effect on promoting Wnt/β-catenin signaling was confirmed by immunoblotting and TOPFlash promoter reporter (Fig. 7a, b). Overexpression of activated β-catenin in MASTL-inhibited cells inhibited apoptosis (40–50%) and levels were similar to controls cells (Fig. 7c) suggesting effects of MASTL on cell viability are mediated by modulating β-catenin expression and activity.

In head and neck cancer, up-regulation of MASTL expression promotes cancer progression and tumor recurrence after initial cancer therapy [2]. In the light of our data that MASTL expression is directly proportional to the CRC progression, we reasoned that MASTL expression may similarly promote resistance against conventional anti-CRC therapy using 5-FU. To determine the validity of this supposition, control, HCT116^MKD, and SW620^MKD cells were subjected to 5FU-treatment (10 or 20 μM). Immunoblotting using cell lysate prepared from these samples demonstrated significant increases in MASTL expression, along with Survivin and Bcl-xL expressions, suggesting a potential role for these molecules in chemotherapeutic resistance (Fig. 8a). Further analysis showed that the increase in Survivin and Bcl-xL expressions in 5FU-treated control cells was significantly reduced in MASTL-inhibited cells even in the presence of 5FU probably making these cells more sensitive to chemotherapy (Fig. 8a). Similar results were obtained in SW620 cells (Additional file 1: Figure S7).

We then hypothesized that inhibition of MASTL would reduce survival signaling downstream of MASTL and induce chemosensitivity. We again subjected control and MASTL knockdown cells to 5FU-treatment (both cell lines being highly metastatic and chemoresistant). Treatment with 5FU could only induce 25–30% cell death in control HCT116 cells. However, 5- FU treatment was significantly more effective in the same cells in the absence of MASTL expression (HCT116^MKD), with cell survival significantly reduced (60–70%) (Fig. 8b, P < 0.001). Similar results were observed in SW620C and SW620^MKD cells. These observations further support a key role for MASTL in resistance to chemotherapeutic agents for colorectal cancer. A postulated model depicting MASTL-dependent regulation of β-catenin to regulate cmyc/Survivin/Bcl-xL expression, associated signaling and cellular functions is presented in Fig. 8c.