TPX2 is a novel prognostic marker for the growth and metastasis of colon cancer

Patient information and tissue specimens

This study was approved by the Institutional Research Ethics Committee and written consents were obtained from all 203 patients with pathologically and clinically confirmed colon cancer. None of the patients had received radiotherapy or chemotherapy before surgery. Staging was based on pathological findings according to the American Joint Committee on Cancer (AJCC). Based on the tumor (T), node (N), and metastasis (M) classification system, we identified 24 cases at stage I, 81 at stage II, 80 at stage III, and 18 at stage IV. The matching adjacent noncancerous tissue, primary colon cancer tissue, and lymph node metastasis lesions from the 203 patients was fixed in formalin and embedded in paraffin for histological analysis and immunohistochemical studies. Fresh samples were dissected manually to remove connective tissues and were immediately stored in liquid nitrogen until western blot analysis.

TMA construction and immunohistochemistry

The tissue array construction procedure has been described previously[19]. Sections (4-μm thick) of TMA slides were prepared and processed for immunostaining. The paraffin sections were de-paraffinized in xylene and rehydrated in a graded alcohol series, boiled with 10 mmol/L of citrate buffer (pH 6) for 10 min, and treated with 0.3% H₂O₂ for 10 min. The steps were performed using the Envision two-step method. The Envision and DAB Color Kit was purchased from Gene Tech Company Limited (Shanghai). The TPX2 anti-human rabbit polyclonal antibody was used at a dilution of 1:200, PBS was used as a negative control. Immunoreactivity was evaluated independently by two researchers in a blinded fashion. The evaluation was based on the staining intensity and extent of staining. The staining intensity was graded as follows, 0: no staining, 1+: mild staining, 2+: moderate staining, and 3+: intense staining. The staining area was scored using the following scale, 0: no staining of cells, 1+: <10% of tissue stained positive, 2+: 10–50% stained positive, and 3+: >50% stained positive. The sum of staining score (intensity + extension) index was designated as follows, 0–2: negative expression, 3–4: weak expression, and 5–6: strong expression.

RNA extraction, reverse transcription (RT), and quantitative real-time PCR (qPCR)

RNA was isolated according to the manufacturer’s instructions (TRIzol, Invitrogen, USA). One microgram of total RNA from each sample was subjected to first-strand cDNA synthesis according to the manufacturer’s recommendations (Promega, USA). Quantitative PCR was performed on a Mastercycler® eprealplex (Eppendorf) with an IQTM SYBR Green Supermix Kit (BIO-RAD) according to the manufacturer’s protocol. TPX2 was amplified with the following primers: 5′-AGGGGCCCTTTGAACTCTTA-3′ (forward primer) and 5′-TGCTCTAAACAAGCCCCATT-3′ (reverse primer). GAPDH was used as an endogenous control with the following primers: 5′-CGGATTTGGTCGTATTGG-3′ (forward primer) and 5′-TCCTGGAAGATGGTGATG-3′ (reverse primer). The cycling conditions for TPX2 and GAPDH were as follows: one cycle at 95°C for 3 min; 40 cycles of 95°C for 15 s, and 60°C for 60 s. The specificity of the PCR amplification was validated by the presence of a single peak in the melting curve analyses. Each RT-qPCR experiment was repeated three times.

Plasmids

For depletion of TPX2, a human siRNA sequence was cloned into the pSilencer™ 2.1-U6 puro Vector (Ambion) according to manufacturer’s protocol. The target sequence was 5′-AAGAATGGAACTGGAGGGCTT-3′. A scrambled siRNA (5′-GTACCGCACGTCATTCGTATC -3′) with no homology to the mammalian mRNA sequences was used as a negative control. Transfection of TPX2-shRNA or control-shRNA plasmid was performed using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.

3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay

Cells were seeded in 96-well plates at an initial density of 0.2 × 10⁴ cells/well. At each time point, cells were stained with 100 μL sterile MTT dye (0.5 mg/mL, Sigma) for 4 h at 37°C, followed by removal of the culture medium and addition of 150 μL of dimethyl sulphoxide (DMSO) (Sigma, St. Louis, MO, USA). The absorbance was measured at 570 nm, with 655 nm as the reference wavelength. All experiments were performed in triplicate.

Cell migration and invasion assays

Cell migration and invasion assays were conducted using a modified 24-well Boyden chamber with a membrane that was uncoated, or coated with Matrigel (BD Biosciences). Briefly, 24 h after transfection of both HCT116 and SW620 cells either with a control (mock or control shRNA) or TPX2 shRNA, the cells were harvested and resuspended in DMEM at a concentration of 5 × 10⁴ cells/mL. Cells prepared in 500 uL of DMEM were loaded in the upper wells, and a medium containing 20% FBS was placed in the lower wells as a chemoattractant stimulus. Cells that had migrated to the bottom surface of the filter were fixed, stained with H&E, and counted under a microscope in three randomly selected fields at a magnification of 200 × .

Gelatin zymography assay

SW620 cells (2 × 10⁵ cells) were seeded in six-well plates and incubated overnight at 37°C. The cells were washed twice with Hanks’ balanced salt solution and cultured for an additional 24 h in serum-free medium. Culture supernatants were collected for collagenase activity assays. Culture supernatants (40 μL) were resolved on a 7.5% sodium dodecyl sulfate polyacrylamide gel that contained 1 mg/mL gelatin (Sigma Chemical Co., St. Louis, MO). The gel was washed for 30 min at room temperature in wash buffer (50.0 mM Tris–HCl [pH 7.5], 15.0 mM CaCl₂, 1.0 μM ZnCl₂, and 2.5% Triton X-100) and then incubated for 24 h at 37°C in the same buffer at a final concentration of 1%. The gel was then stained with 0.1% Coomassie Brilliant Blue R-250; clear zones against the blue background indicated the presence of gelatinolytic (i.e., collagenase) activity.

Soft agar assay

Cells were suspended in 0.3% agar medium (DMEM containing 10% FBS) and then plated on a 0.6% agar base layer at a concentration of 1 × 10³ cells per six-well plate. The cells were incubated in a humidified atmosphere (5% CO₂) at 37°C for 10 days, following which the number of colonies that were 50-μm or larger were counted.

Xenografted tumor model

SW620 cancer cells with stably silenced TPX2 (sh-TPX2) or control (sh-Control and mock) (1 × 10⁶ cells) were subcutaneously injected into the flanks of BALB/c-nu mice as previously described[20]. All procedures involving mice were conducted in accordance with Fudan University Shanghai Cancer Center Animal Care guidelines. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize possible alternatives to in vivo techniques.

Statistics

ANOVA test was used to determine the statistical significance of differences between experimental groups. The Kaplan-Meier method was used to analyze colon cancer patients’ cumulative survival rate. A Cox proportional hazards model was used to calculate univariate and multivariate hazard ratios for the study variables. SPSS software 13.0 (SPSS, Chicago, IL, USA) was used for the analyses. A P value of 0.05 was considered as statistically significant.

Aberrant overexpression of TPX2 in colon cancer tissue and cell lines

Real-time PCR analyses revealed that mRNA expression level of TPX2 was markedly higher in all colon cancer cell lines than in non-malignant human NCM460 colonic cell line (Figure 1A1). And the protein expression level of TPX2 was also higher in the colon cancer cell lines but not so markedly as its mRNA expression level (Figure 1A2). Furthermore, comparative analysis showed that the mRNA and protein levels of TPX2 were differentially upregulated in all 4 colon cancer samples compared to the matched adjacent non-tumor tissues (Figure 1B1, B2), suggesting that TPX2 expression is upregulated in colon cancer. The clinicopathologic characteristics of four patients used in western Blot and RT-PCR analysis was provided in the (Additional file1: Table S1).

Association between TPX2 expression and the clinical features of colon cancer

To determine whether TPX2 clinically correlated with colon cancer progression, the expression of TPX2 was determined by immunohistochemistry in a tissue microarray containing 203 cases of primary colon cancer paired with their non-cancerous tissue and 66 lymph node metastases (LNM). We found that TPX2 was dramatically upregulated in primary colon cancer (61.1%), but it was either only detected minimally, or not at all (98%) in adjacent normal colonic tissue. The representative expression pattern in both tumor and non-tumor samples are shown in Figure 2A. The quantitative analysis of IHC staining is summarized in Table 1. We observed that the expression levels of TPX2 were closely correlated with the T classification (P = 0.008), lymph node involvement (P = 0.001), distant metastasis (P = 0.048), and clinical stage (P = 0.004) in colon cancer patients. Collectively, these data indicate that TPX2 may be involved in colon cancer carcinogenesis and metastasis.

TPX2 expression is significantly associated with lymph node metastasis and poor survival in colon cancer patients

Furthermore, we postoperatively analyzed the predictive significance of TPX2 in the development of distant metastasis. The metastasis-free survival (MFS) time was analyzed in 185 patients in stages I-III, who accepted radical colectomy. The proportion of patients who developed metastasis from primary colon cancer after radical colectomy differed substantially between the TPX2-positive and TPX2-negative group (Figure 2B, left). The risk of developing distant metastases after radical colectomy was much higher in patients with a TPX2-positive tumor relative to patients with a TPX2-negative tumor (P < 0.001) (TPX2-positive, 40 [36.7%] of 109 patients, mean follow-up 64.5 months [range 58.4-70.6]; TPX2-negative, 8 [10.5%] of 76, 81.2 [77.3-85.1]). Based on these results, TPX2 could serve as a novel prognostic marker to predict risk of distant metastases in patients with radical colectomy (Hazard Ratio, 4.3; [95% CI 2.0-9.2]; P < 0.001).

Downregulation of TPX2 inhibits proliferation of colon cancer cells in vitro and in vivo

The impact of TPX2 on proliferation of colon cancer cells was evaluated by knockdown of TPX2. The MTT assay showed that depletion of TPX2 expression caused a marked reduction in the viability of HCT116 and SW620 cells (Figure 3A and B). These results demonstrate that TPX2 suppression could inhibit the proliferation ability of colon cancer cells.

Since TPX2 was correlated with the clinical characteristics of colon cancer, we further investigated the effect of TPX2 on the tumorigenic activity of colon cancer cell lines. Control cells (MOCK and shRNA) and SW620-TPX2-shRNA cells were subcutaneously injected into nude mouse (n = 5/group). As shown in Figure 3C and D, the tumors formed from SW620-TPX2-shRNA cells grew much more slowly than those from the control cells. After 4 weeks, the weight of tumors induced by the TPX2-suppressed cells was significantly reduced when compared to that induced by control cells (Figure 3E). Furthermore, IHC-stained sections of mouse tumors that demonstrated a high level of TPX2 also strongly stained for Ki67 (Figure 3F), consistent with the cell proliferation results in vitro. Together, our results indicated that TPX2 plays a critical role in the tumorigenicity of colon cancer cell lines both in vitro and in vivo.

Gene Silencing of TPX2 expression in colon cancer cells leads to Akt reduction

As TPX2 expression is linked to poor survival of colon cancer patients, we wanted to further explore the molecular mechanism of its action. We found that the phosphorylation and activation of Akt was markedly reduced in shRNA-TPX2 transfected cells compared with the control group, while downregulation of TPX2 did not affect ERK-1/2 activation, which are involved in a different pathway from Akt (Figure 4A). Furthermore, knocking down TPX2 in SW620 reduced nuclear Akt (Figure 4E).

To confirm whether TPX2 induced proliferation of colon cancer cells through the Akt pathway, we overexpressed TPX2 in SW480, which is a lower grade colon cancer cell line, then treated with a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Blockade of Akt activation suppressed the proliferation induced by TPX2 in SW480 cells, as determined by a colony formation assay and MTT assay (Figure 4B, C and D). Together, these data suggest that downregulation of TPX2 inhibits Akt activation, and Akt activation is an important step in the TPX2-induced proliferation of colon cancer cells.

Gene silencing of TPX2 suppresses the migratory and invasive ability of colon cancer cells through a modulation of MMP2 expression and activity

As TPX2 is linked to the advanced clinical stage and poorer MFS of colon cancer patients, we then wanted to determine the possible role of TPX2 on cell migration and invasion activity in vitro. The effect of TPX2 knockdown on migration potency of SW620 cells was assayed using migration chambers. Compared to the control groups, TPX2 silencing resulted in significantly reduced migratory ability (P <0.01, Figure 5A and B). We also assessed the effect of TPX2 depletion on tumor invasion and demonstrated that disruption of endogenous TPX2 expression also attenuated cell invasive potential in colon cancer cells (Figure 5A and B). The results indicate a critical role of TPX2 in the metastasis of colon cancer.

To better understand the role of TPX2 in the progression and metastasis of colon cancer cells, we explored the possible roles of metastasis-related molecules downstream of TPX2. We found that knockdown of endogenous TPX2 led to substantial reduction in both mRNA and protein level of MMP2 (Figure 6A, B, and D). We next examined the potential effect of TPX2 on the activity of MMP2 using zymography analysis. Higher activity of MMP2 was observed in control group compared to ShRNA-TPX2 treated cells (Figure 6C). The data suggest that TXP2 may be a potential target in colon cancer therapy due to its ability to modulate downstream MMP2 expression and activity.