Interferon-γ in the tumor microenvironment promotes the expression of B7H4 in colorectal cancer cells, thereby inhibiting cytotoxic T cells.

The bioactivity of interferon-γ (IFN-γ) in cancer cells in the tumor microenvironment (TME) is not well understood in the current immunotherapy era. We found that IFN-γ has an immunosuppressive effect on colorectal cancer (CRC) cells. The tumor volume in immunocompetent mice was significantly increased after subcutaneous implantation of murine CRC cells followed by IFN-γ stimulation, and RNA sequencing showed high expression of B7 homologous protein 4 (B7H4) in these tumors. B7H4 promotes CRC cell growth by inhibiting the release of granzyme B (GzmB) from CD8+ T cells and accelerating apoptosis in CD8+ T cells. Furthermore, interferon regulatory factor 1 (IRF1), which binds to the B7H4 promoter, is positively associated with IFN-γ stimulation-induced expression of B7H4. The clinical outcome of patients with CRC was negatively related to the high expression of B7H4 in cancer cells or low expression of CD8 in the microenvironment. Therefore, B7H4 is a biomarker of poor prognosis in CRC patients, and interference with the IFN-γ/IRF1/B7H4 axis might be a novel immunotherapeutic method to restore the cytotoxic killing of CRC cells.

Cdc42 subcellular relocation in response to VEGF/NRP1 engagement is associated with the poor prognosis of colorectal cancer.

Microscopic indications of malignancy and hallmark molecules of cancer are pivotal to determining cancer patient prognosis and subsequent medical intervention. Here, we found that compared to apical expression of Cdc42, which indicated that basal expression of Cdc42 occurred at the migrating cell front, glandular basal expression of Cdc42 (cell division cycle 42) in tissues indicated poorer prognoses for colorectal cancer (CRC) patients. The current study shows that activated Cdc42 was rapidly recruited to the migrating CRC cell front after VEGF stimulation through engagement of membrane-anchored neuropilin-1 (NRP1). When VEGF signalling was blocked with NRP1 knockdown or ATWLPPR (A7R, antagonist of VEGF/NRP1 interaction), Cdc42 activation and relocation to the cell front was attenuated, and filopodia and invadopodia formation was inhibited. The VEGF/NRP1 axis regulates directional migration, invasion, and metastasis through Cdc42 activation and relocation resulting from actin filament polymerisation of the extensions of membrane protrusions. Collectively, the immuno-micromorphological pattern of subcellular Cdc42 at the cell front indicated aggressive behaviours and predicted poor prognosis in CRC patients. Disruption of the intra- and extracellular interactions of the VEGF/NRP1 axis or Cdc42 relocation could be performed in clinical practice because it might inhibit cancer cell motility and metastasis.

High expression of PRKDC promotes breast cancer cell growth via p38 MAPK signaling and is associated with poor survival.

BACKGROUND: DNA-Dependent Protein Kinase Catalytic Subunit (PRKDC), a key component of the DNA damage repair pathway, is associated with chemotherapy resistance and tumor progression. METHODS: Here we analyzed transcriptome data of ~2,000 breast cancer patients and performed functional studies in vitro to investigate the function of PRKDC in breast cancer. RESULTS: Our results revealed overexpression of PRKDC in multiple breast cancer subtypes. Consistent with patients' data, overexpression of PRKDC was also observed in breast cancer cell lines compared to normal breast epithelial cells. Knockdown of PRKDC in MCF-7 and T47D breast cancer cell lines resulted in proliferation inhibition, reduced colony formation and G2/M cell cycle arrest. Furthermore, we showed that PRKDC knockdown induced proliferation inhibition through activation of p38 MAPK, but not ERK MAPK, signaling pathway in breast cancer cells. Blockage of p38 MAPK signaling could largely rescue proliferation inhibition and cell cycle arrest induced by PRKDC knockdown. Moreover, we analyzed gene expression and clinical data from six independent breast cancer cohorts containing ~1,000 patients. In all cohorts, our results consistently showed that high expression of PRKDC was significantly associated with poor survival in both treated and untreated breast cancer patients. CONCLUSION: Together, our results suggest that high expression of PRKDC facilitates breast cancer cell growth via regulation of p38 MAPK signaling, and is a prognostic marker for poor survival in breast cancer patients.

PRKDC is a prognostic marker for poor survival in gastric cancer patients and regulates DNA damage response.

A hallmark of gastric cancer is the high rate of genomic instability associated with deregulation of DNA damage repair pathways. DNA-Dependent Protein Kinase Catalytic Subunit (PRKDC) is a key component of the non-homologous end-joining (NHEJ) pathway. By reanalyzing transcriptome data of 80 pairs of gastric cancer tumors and the adjacent normal tissues from non-treated patients, we identified PRKDC as the top upregulated DNA damage repair genes in gastric cancer. High expression of PRKDC is associated with poor survival of gastric cancer patients, and genomic amplification of the gene is frequently observed across most gastric cancer subtypes. Knockdown of PRKDC in gastric cell lines resulted in reduced proliferation and cell cycle arrest. Furthermore, we showed that loss of PRKDC induced DNA damage and enhanced gastric cancer cell chemosensitivity to DNA-damaging reagents. Together, our results suggest that PRKDC is a prognostic marker of poor survival and is a putative target to overcome chemoresistance in gastric cancer.

Prohibitin, relocated to the front ends, can control the migration directionality of colorectal cancer cells.

Directional migration is a cost-effective movement allowing invasion and metastatic spread of cancer cells. Although migration related to cytoskeletal assembly and microenvironmental chemotaxis has been elucidated, little is known about interaction between extracellular and intracellular molecules for controlling the migrational directionality. A polarized expression of prohibitin (PHB) in the front ends of CRC cells favors metastasis and is correlated with poor prognosis for 545 CRC patients. A high level of vascular endothelial growth factor (VEGF) in the interstitial tissue of CRC patients is associated with metastasis. VEGF bound to its receptor, neuropilin-1, can stimulate the activation of cell division cycle 42, which recruits intra-mitochondrial PHB to the front end of a CRC cell. This intracellular relocation of PHB results in the polymerization and reorganization of filament actin extending to the front end of the cell. As a result, the migration directionality of CRC cells is targeted towards VEGF. Together, these findings identify PHB as a key modulator of directional migration of CRC cells and a target for metastasis.

Myofibrotic malformation vessels: unique angiodysplasia toward the progression of hemorrhoidal disease.

BACKGROUND: The etiology and pathogenesis of hemorrhoids is unclear, although hemorrhoids are a worldwide disease in men and women, with peak prevalence at 45-65 years of age. Hemorrhoidal cushions as the anal venous plexi are normal anatomical structures from infancy. This study attempts to reveal the angiodysplasia and other pathological changes in association with different degrees of symptomatic hemorrhoids. MATERIALS AND METHODS: A total of 281 patients with internal hemorrhoids from degree I to IV underwent hemorrhoidectomy. The vascular changes were analyzed by microscopic assessment and software analysis, with Masson's trichrome, CD34, and smooth muscle actin. RESULTS: The hemorrhoidal tissues exhibited abnormal vessels in the mucosae and submucosae that we termed them as myofibrotic malformation vessels (MMVs). MMVs are not ascribed to arteries or veins because they exhibit enlarged and tortuous lumens with smooth muscle dysplasia and fibrotic deposition in the walls without overlying mucosal ulceration. The muscularis mucosae also showed smooth muscle dysplasia and fibrosis, even if it were interrupted by the intruding MMVs. The statistical data indicated that the severity of all the changes correlate positively with the progression of hemorrhoids (P<0.001). Hemorrhoidal patients are prone for reoccurrence even with prolapsing hemorrhoid when compared with the conventional hemorrhoidectomy. Multiple logistic regression analysis showed that MMVs in mucosal propria, mean thickness of mucosal muscularis layer, and fibrotic changes in MMV were independent risk factors for MMVs in hemorrhoidal disease. CONCLUSION: MMVs and muscularis mucosae dysplasia reciprocally contribute to hemorrhoidal exacerbation. The novel findings of this study propose that the characteristic features of MMVs and muscularis mucosae dysplasia of the anorectal tube ultimately cause symptomatic hemorrhoids, which could affect the clinical management of hemorrhoidal disease through the use of surgery to target the malformed vessels.

MIF, secreted by human hepatic sinusoidal endothelial cells, promotes chemotaxis and outgrowth of colorectal cancer in liver prometastasis.

Growth and invasion of metastatic colorectal cancer (CRC) cells in the liver depend on microenvironment. Here, we showed that human hepatic sinusoidal endothelial cells (HHSECs) induce chemotaxis and outgrowth of CRC cells. Macrophage migration inhibitory factor (MIF), released by HHSECs, stimulated chemotaxis of CRC cells. MIF secreted by HHSECs, but not by CRC cells themselves, promoted migration and epithelial-mesenchymal transition (EMT) and facilitated proliferation and apoptotic resistance of CRC cells. In orthotopic implantation models in nude mice, exogenous MIF stimulated growth of CRC cells and metastasis. Furthermore, MIF accelerated mobility of CRC cells by suppressing F-actin depolymerization and phosphorylating cofilin. Noteworthy, MIF levels were correlated with the size of hepatic metastases. We suggest that HHSECs and paracrine MIF promote initial migration and proliferation of CRC cells in the hepatic sinusoids to generate liver metastases.

Interaction between Oc-1 and Lmx1a promotes ventral midbrain dopamine neural stem cells differentiation into dopamine neurons.

Recent studies have shown that Onecut (Oc) transcription factors may be involved in the early development of midbrain dopaminergic neurons (mdDA). The expression profile of Oc factors matches that of Lmx1a, an important intrinsic transcription factor in the development of mDA neuron. Moreover, the Wnt1-Lmx1a pathway controls the mdDA differentiation. However, their expression dynamics and molecular mechanisms remain to be determined. To address these issues, we hypothesize that cross-talk between Oc-1 and Lmx1a regulates the mdDA specification and differentiation through the canonical Wnt-ß-catenin pathway. We found that Oc-1 and Lmx1a displayed a very similar expression profile from embryonic to adult ventral midbrain (VM) tissues. Oc-1 regulated the proliferation and differentiation of ventral midbrain neural stem cells (vmNSCs). Downregulation of Oc-1 decreased both transcript and protein level of Lmx1a. Oc-1 interacted with lmx1a in vmNSCs in vitro and in VM tissues in vivo. Knockdown of Lmx1a reduced the expression of Oc-1 and Wnt1 in vmNSCs. Inhibiting Wnt1 signaling in vmNSCs provoked similar responses. Our data suggested that Oc-1 interacts with Lmx1a to promote vmNSCs differentiation into dopamine neuron through Wnt1-Lmx1a pathway.

Metastatic cancer stem cells: from the concept to therapeutics.

Metastatic cancer stem cells (MCSCs) refer to a subpopulation of cancer cells with both stem cell properties and invasion capabilities that contribute to cancer metastasis. MCSCs have capability of self-renewal, potentials of multiple differentiation and development and/or reconstruction of cancer tissues. As compared with stationary cancer stem cells, MCSCs are capable of invasion to normal tissues such as vasculatures, resistance to chemo- and/or radio-therapies, escape from immune surveillance, survival in circulation and formation of metastasis. MCSCs are derived from invasive cancer stem cells (iCSCs) due to the plasticity of cancer stem cells, which is one of the characteristics of cancer cell heterogeneity. Both stages of iCSCs and MSCSs are the potential therapeutic targets for cancer metastasis in the future strategies of personalized cancer therapy.

CLIC4, ERp29, and Smac/DIABLO derived from metastatic cancer stem-like cells stratify prognostic risks of colorectal cancer.

PURPOSE: Cancer stem-like cells have been well accepted to be involved in recurrence and metastasis of cancers, but the prognostic potential of biomarkers integrating with metastasis and cancer stem-like cells for colorectal cancer is unclear. EXPERIMENTAL DESIGN: We identified three proteins, CLIC4, ERp29, and Smac/DIABLO, from metastatic cancer stem-like cells of colorectal cancer and verified the proteins' role in metastatic behaviors. The proteins were detected by IHC in colorectal cancer tumors and matched colonic mucosa from patients with colorectal cancer who underwent radical surgery in the training cohort. The associations between proteins expression levels and five-year disease-specific survival (DSS) were evaluated to predict the survival probability in the training cohort of 421 cases and the validation cohort of 228 cases. RESULTS: A three-protein panel including CLIC4, ERp29, and Smac/DIABLO, which was generated from multivariate analysis by excluding clinicopathologic characteristics from the training cohort, distinguished patients with colorectal cancer into very low-, low-, middle-, and high-risk groups with significant differences in five-year DSS probability (88.6%, 63.3%, 30.4%, 11.4%; P < 0.001). The panel is independent from tumor-node-metastasis staging system and histologic grading to predict prognosis, and also enables classification of validation cohort into four risk stratifications (five-year DSS probability is 98.2%, 80.2%, 25.6%, and 2.7%; P < 0.001). CONCLUSIONS: CLIC4, ERp29, and Smac/DIABLO integrated into a novel panel based on cancer stem-like cells in association with metastasis stratify the prognostic risks of colorectal cancer. Prediction of risks with molecular markers will benefit clinicians to make decisions of individual management with postoperative colorectal cancer patients.

A modified method for isolation of bladder cancer stem cells from a MB49 murine cell line.

BACKGROUND: The vaccine was efficiently effective against bladder cancer in earlier studies. However, a part of the mouse bladder tumour regrew due to regression after a period of time as the cancer stem cells could not be eliminated. In this study, we showed a modified method for the isolation of MB49 bladder cancer stem cells (MCSCs). METHODS: Through a comparison of different serum-free culture mediums (SFM), MCSCs were isolated by a combination of the limited dilution method and the optimal SFM method. The characterizations of MCSCs were verified by the fluorescence activated cell sorting, the quantitative polymerase chain reaction, the western blotting, the cell proliferation assay, the soft agar assay, the transwell assay, the resistance to chemotherapy assay and the tumor xenograft formation assay. RESULTS: The optimal SFM contained a RPMI1640+ epidermal growth factor (20 ng/ml), a basic fibroblast growth factor (20 ng/ml), a leukemia inhibitory factor (20 ng/ml), a B-27 serum-free supplement (20 µl/ml), and a bovine serum albumin (4 µg/ml). MCSCs possessed the high expression of cancer stem cell markers (CD133, CD44, OCT4, NANOG, and ABCG2) and the ability of differentiation. In functional comparisons, MCSCs had higher proliferative abilities, lower susceptibility to chemotherapy, greater migration in vitro, and stronger tumorigenic abilities in vivo. CONCLUSION: MCSCs displayed specific cancer stem cells properties. Our study showed MCSCs were isolated successfully with a modified method using a combination of limited dilution and SFM methods.

Perfusion heterogeneity in breast tumors for assessment of angiogenesis.

OBJECTIVES: The purpose of this study was to investigate the perfusion heterogeneity of malignant and benign breast tumors and assay their vascular architecture changes and molecular expression, thereby evaluating the relevance between imaging and histologic characteristics of angiogenesis. METHODS: Real-time grayscale contrast-enhanced sonography was performed in 310 women with 317 breast tumors. The enhancement patterns and perfusion parameters for malignant and benign tumors were analyzed by contrast-enhanced sonography with microvascular imaging and quantitative time-intensity curve analysis. Structural characteristics were observed by light and electron microscopy. The microvessel density, vascular endothelial growth factor (VEGF) expression, and human kinase insert domain-containing receptor (KDR) expression for all tumors were assessed by immunohistochemical staining of CD31, KDR, and VEGF. RESULTS: Surgical pathologic analysis showed 163 malignant and 154 benign tumors. Significant morphologic differences, including perfusion defects, vessel distortion, vessel dilatation, and heterogeneous enhancement, were observed between the malignant and benign groups (P < .05). The mean perfusion parameters (peak intensity, ascending slope, area under the curve, and wash-out time) were greater in the malignant tumors (P < .05). There were significant differences in the peak intensity, ascending slope, area under the curve, and wash-out time between peripheral and central regions of the malignant tumors (P < .05) but none in the benign tumors. Vessels had various morphologic and distributional characteristics in the peripheral and central regions of the malignant tumors. The microvessel density and VEGF and KDR expression were significantly higher in the malignant group (P < .05), especially in the peripheral regions. CONCLUSIONS: Perfusion heterogeneity was closely associated with the tumor microvascular architecture and molecular expression. Perfusion features, especially regional morphologic and hemodynamic features, can provide valuable information for differentiating malignant from benign breast tumors.

MR imaging of cerebral extraventricular neurocytoma: a report of 9 cases.

Extraventricular neurocytoma is a rare entity, most frequently occurring in brain parenchyma outside the ventricular system. The purpose of this study was to characterize the MR imaging findings in a series of 9 patients with EVN verified by results of pathologic examination. All 9 EVNs were solitary and intracranially located. Eight lesions were well demarcated, and 3 showed intratumoral hemorrhage. The solid parts of 7 tumors were primarily isointense on T1-weighted images and heterogeneously enhanced on T1WI with contrast. Although cerebral EVNs can present a wide spectrum of appearances on MR, the imaging patterns appear to vary according to anatomic location and cellularity. Lesions in frontal or parietal lobes often present as well-demarcated large masses with cystic degeneration, hemorrhage, mild-to-moderate edema, and inhomogeneous enhancement. Moreover, the general isointensity of the solid parts of EVN on T1WI may be of some specificity.

The establishment of supramolecular immunobead real-time PCR and the identification of Cox-2 as a metastasis-related marker in colorectal carcinoma.

Cyclooxygenase-2 (Cox-2) is an inducible enzyme that converts arachidonic acid to prostaglandins, and it is hypothesized to induce carcinogenesis and metastasis in colorectal cancer. Our previous data also indicated that a higher expression level of Cox-2 was correlated with colorectal cancer metastasis. The Cox-2 protein was detected in the glandular cavity of colorectal cancer and the surrounding interstitial tissues. The usefulness of the Cox-2 gene as a gene therapy target and diagnostic marker remains unknown. In this study, a method using immuno-PCR and real-time PCR followed by supramolecular immunobead real-time PCR was established and used to detect the expression of Cox-2 in serum samples of nude mice with colorectal carcinoma. In addition, we established a Cox-2 gene stable knockdown colorectal cell line (SW480-EGFP-Cox-2 shRNA) using lentiviral vector-mediated RNA interference (RNAi) technology and established an imageable colorectal cancer metastasis mouse model. We found that the proliferation, invasion and tumorigenesis of SW480-EGFP-Cox-2 shRNA cells were attenuated compared with SW480 cells. In vivo experiments also demonstrated that angiogenesis in the Cox-2 knockdown colorectal cancer cells was decreased. The whole body optical imaging revealed that the SW480-EGFP-Cox-2 shRNA cells had an abrogated ability to develop metastases in the lymph nodes, lungs or liver in vivo. The improved immunobead PCR assay detected significantly lower Cox-2 protein levels in the serum samples of the SW480-EGFP-Cox-2 shRNA group compared with those of the SW480-EGFP-Cox-2-Ctrl shRNA group. In conclusion, our results indicated that the knockdown of Cox-2 expression suppressed the proliferation and invasion of colorectal cancer cells both in vitro and in vivo. This study also demonstrated that silencing Cox-2 in vivo reduced the metastastic potential of colorectal cancer. Thus, Cox-2 is a promising marker for the diagnosis of colorectal metastasis and a potential therapeutic target for colorectal cancer.

[Infantile DiGeorge syndrome: autopsy diagnosis and clinicopathologic analysis in 5 cases].

OBJECTIVE: To investigate clinicopathological features of DiGeorge syndrome (DGS). METHOD: The clinical features, histological and immunohistochemical findings were analyzed in 5 cases of DGS by autopsy. RESULTS: Five cases of DGS in male infants aged 4 days, 1 month, 7 months, 10 months, and 13 months respectively. Gross and microscopic observations revealed that thymic cortex was depleted of lymphocytes or showed few, dispersed lymphocytes. The thymic medulla showed predominantly epithelial cells with calcified Hassall bodies as well as lymphocyte depletion. T lymphocytes were also scarce in the tonsils, lymph nodes, spleen, and mucosa-associated lymphatic tissue of ileum. In addition, 3 of the 5 patients also showed parathyroid aplasia or dysplasia, and congenital hypertrophy of the ventricular septum. CONCLUSIONS: The pathological changes indicate that clinicians should be aware of defects of immune system if the infants suffer from severe infections. Pathologists should recognize the importance of abnormalities of lymphohematopoietic tissues in the diagnosis of primary immunodeficiency diseases such as DGS.

[Construction of a PCR chip for detecting genes associated with metastatic colorectal cancer and its preliminary application].

OBJECTIVE: To construct a PCR chip with a gene panel for predicting and diagnosing metastatic colorectal cancer. METHODS: The PCR chip was constructed by integrating 29 genes related to colorectal cancer metastasis identified by gene chip analysis and 3 housekeeping genes into a gene panel. The PCR chip was used for detecting the mRNA expressions of the integrated genes in colorectal cell lines, cancerous specimen and adjacent normal mucosa. The primers for amplification were refined and optimized by several rounds of preliminary reactions. RESULTS: The PCR chip containing the 29 candidate genes and 3 housekeeping genes was successfully constructed, which showed specific amplifications of the genes. The results of the PCR chip for detecting the mRNA of the 29 genes related to colorectal cancer metastasis showed a concordance rate of 86% (25 out of 29) with the gene chip data. Application of the PCR chip in the examination of the clinical specimens identified 15 differentially expressed genes between metastatic colorectal cancer and colorectal cancer without metastasis. CONCLUSION: The constructed PCR chip is reliable in the prediction of metastasis of colorectal cancer, and provides a molecular means for evaluating the prognosis of colorectal cancer metastasis.

[Application of 99mTc-SPECT-CT and carbon nanoparticles suspension injection in sentinel lymph node mapping for rectal cancer].

OBJECTIVE: To evaluate the accuracy of sentinel lymph node mapping(SLM) in patients with rectal cancer by single-photon emission computed tomography (SPECT-CT) lymphoscintigraphy and carbon nanoparticles suspension injection. METHODS: Twelve patients with clinical T(1-2)N(0)M(0) rectal cancer were selected and locally injected with technetium-(99m)sulfur-colloid and carbon nanoparticles suspension by endoscope one day before surgery, followed by SPECT-CT scanning 1, 3 and 5 hours later. Radioactive isotope(RI) uptake of each sentinel node(SN) basin with location preoperatively determined by SPECT-CT was postoperatively calculated using gamma probe. Nodes with the highest RI uptake, the number of which was also pre-determined by SPECT-CT, was defined as SNs. Immunohistochemical cytokeratin staining was performed for all the SNs and non-SNs. RESULTS: The rate of sentinel node detection was 91.7%(11/12) with at least one SN(1-3) per patient. Ten cases showed metastasis-negative in SNs as well as all the resected regional nodes by immunohistochemical cytokeratin staining. Only one patient had positive nodes in both SN and non-SNs. The accuracy of SLM was 100%. CONCLUSION: SPECT-CT lymphoscintigraphy and carbon nanoparticles suspension injection can effectively detect the anatomic location and number of sentinel nodes, and improve the accuracy of SLM for rectal cancer.

HOXB7 as a prognostic factor and mediator of colorectal cancer progression.

PURPOSE: This study was to investigate the clinicopathologic significance and potential role of HOXB7 in the development and progression of colorectal cancer (CRC). EXPERIMENTAL DESIGN: The relationship between HOXB7 expression and clinical characteristics of CRC was analyzed in 224 paraffin-embedded archived CRC specimens by immunohistochemistry (IHC). The effects of HOXB7 on cell growth and proliferation, as well as on tumorigenesis, were examined both in vitro and in vivo, using MTT assay, colony formation assay, cell cycle analysis, soft agar assay, and tumorigenesis in nude mice. Western blotting and real-time reverse transcriptase-PCR were performed to examine the impact of HOXB7 on the PI3K/Akt and MAPK signaling pathways. RESULTS: HOXB7 protein level was significantly correlated with advanced Dukes stage (P < 0.001), T stage (P = 0.012), distant metastasis (P = 0.042), higher proliferation index (P = 0.007) and poor survival of patients (P = 0.005). Enforced expression of HOXB7 in CRC cell lines significantly enhanced cell growth, proliferation and tumorigenesis. Conversely, knockdown of HOXB7 caused an inhibition of cell growth, proliferation, and tumorigenesis. We also showed that HOXB7 accelerated G(0)-G(1) to S-phase transition concomitantly with upregulation of cyclin D1 and downregulation of p27Kip1. On the contrary, knockdown of HOXB7 caused G(1)-S-phase arrest, downregulation of cyclin D1 and upregulation of p27Kip1. Enforced expression of HOXB7 could enhance PI3K/AKT and MAPK pathway activity. CONCLUSION: Our findings suggest that HOXB7 protein, as a valuable marker of CRC prognosis, plays an important role in the development and progression of human CRC.

FMNL2 is a positive regulator of cell motility and metastasis in colorectal carcinoma.

FMNL2 is a member of diaphanous-related formins which act as effectors of Rho family GTPases and control the actin-dependent processes such as cell motility or invasion. We previously found that FMNL2 overexpression in metastatic cell lines and tissues of colorectal carcinoma is associated with more aggressive tumour behaviour. Here we used gain-of-function and loss-of-function approaches to investigate the effects of FMNL2 on colorectal carcinoma in vitro and in vivo. Forced expression of FMNL2 caused a significant increase in tumour cell proliferation, motility, invasion in vitro, and metastasis in vivo, whereas FMNL2 depletion showed opposite effects. We examined gene expression profiles following knockdown of FMNL2 in SW480/M5 cells. Expression of 323 genes was up-regulated by more than two-fold, whereas 222 genes were down-regulated by more than two-fold in FMNL2-depleting SW480/M5 cells. Gene ontology analysis showed that most of genes belong to functional categories such as cell cycle, cytoskeleton, transcription factor, and G-protein modulator. Pathway analysis revealed that cytoskeletal regulation by the Rho GTPase pathway, the Wnt pathway, the G-protein pathway, and the P53 pathway were affected by FMNL2. Many of these genes are in functional networks associated with cell proliferation, metastasis, Wnt or the Rho signalling pathway involved in the regulation of FMNL2. The expression of five differentially expressed genes including CXXC4, CD200, VAV1, CSF1, and EPHA2 was validated by real-time PCR and western blot analysis. Thus, FMNL2 is a positive regulator of cell motility, invasion, and metastasis of colorectal carcinoma.