NUBPL,a novel metastasis‐related gene,promotes colorectal carcinoma cell motility by inducing epithelial–mesenchymal transition

Nucleotide binding protein‐like, NUBPL, is an assembly factor for human mitochondrial complex I, which is the biggest member of the mitochondrial respiratory chain. However, the relationship between NUBPL and carcinoma progression remains unknown. In this study, NUBPL was characterized for its role in colorectal cancer (CRC) and the underlying molecular mechanisms. Data (n = 197) from the Oncomine database revealed that mRNA levels of NUBPL were remarkably overexpressed in CRC tissues compared with normal tissues. In addition, immunohistochemical analysis of 75 pairs of CRC and non‐tumor tissues showed that the expression level of NUBPL was significantly higher in CRC tissues, and its expression level was positively associated with lymph node metastasis (P = 0.028) and advanced staging (P = 0.030). Expression of NUBPL in metastatic lymph nodes of CRC patients was also detected by immunohistochemical staining and high expression levels of NUBPL were observed. Overexpression of NUBPL significantly promoted the migration and invasion ability of CRC cell lines SW480 and SW620, whereas knockdown of NUBPL lead to an opposite effect. Our further study found that NUBPL could induce epithelial–mesenchymal transition (EMT), characterized by downregulation of epithelial markers (E‐cadherin) and upregulation of mesenchymal markers (N‐cadherin and vimentin). Moreover, NUBPL was able to activate ERK, which is believed to promote EMT and tumor metastasis. Inhibition of ERK suppressed the NUBPL‐induced changes in EMT and cell motility. These data showed that NUBPL plays a vital role in CRC migration and invasion by inducing EMT and activating ERK. It might be a novel therapeutic target for CRC.     

DCLK1 promotes epithelial‐mesenchymal transition via the PI3K/Akt/NF‐κB pathway in colorectal cancer

Double cortin‐like kinase 1 (DCLK1) plays important roles during the epithelial‐mesenchymal transition (EMT) process in human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF‐κB‐dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF‐κBp65 subunit expression through the PI3K/Akt/Sp1 axis and activated NF‐κBp65 through the PI3K/Akt/IκBα pathway during the EMT of CRC cells. Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collectively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential therapeutic target for CRC metastasis.     

Constitutively active MEK1 is sufficient to induce epithelial‐to‐mesenchymal transition in intestinal epithelial cells and to promote tumor invasion and metastasis

Constitutive activation of the MAP kinase kinase MEK1 induces oncogenic transformation in intestinal epithelial cells. Loss of cell–cell adhesion followed by the dissociation of epithelial structures is a prerequisite for increased cell motility and tumor invasion. This phenotypic switch is designated epithelial‐to‐mesenchymal transition (EMT). EMT also plays an important role in determining the dissemination of tumors. However, the role of MEK1 in intestinal EMT, tumor invasion and metastasis has not been elucidated. To determine the functions of activated MEK1 in intestinal tumorigenesis, we established intestinal epithelial cell lines that overexpress wild‐type MEK1 (wtMEK) or activated MEK1 (caMEK). Our results indicate that expression of caMEK is sufficient to induce EMT as confirmed with the induction of N‐cadherin, vimentin, Snail1 and Snail2, whereas a reduction in E‐cadherin, occludin, ZO‐1 and cortical F‐actin was noted. The Snail1 and Snail2 promoter analyses revealed that Egr‐1 and Fra‐1, an AP‐1 protein, are responsible for MEK1‐induced Snail1 and Snail2 expression, respectively. Cells expressing activated MEK1 clearly acquired an invasive capacity when compared to wtMEK‐expressing cells. Zymography studies confirmed elevated levels of MMP2 and MMP9 activities in media of caMEK‐expressing cells. Importantly, cells expressing activated MEK1 induced tumors with short latency in correlation with their ability to induce experimental metastasis in vivo and to express factors known to promote colorectal cancer cell metastasis. In conclusion, our results demonstrate, for the first time, that constitutive activation of MEK1 in intestinal epithelial cells is sufficient to induce an EMT associated with tumor invasion and metastasis. © 2009 UICC     

Polycystin‐1 and polycystin‐2 are involved in the acquisition of aggressive phenotypes in colorectal cancer

The polycystins PC1 and PC2 are emerging as major players in mechanotransduction, a process that influences all steps of the invasion/metastasis cascade. We hypothesized that PC1 and PC2 facilitate cancer aggressiveness. Immunoblotting, RT‐PCR, semi‐quantitative and quantitative real‐time PCR and FACS analyses were employed to investigate the effect of polycystin overexpression in colorectal cancer (CRC) cells. The impact of PC1 inhibition on cancer‐cell proliferation was evaluated through an MTT assay. In vitro data were analyzed by Student's t‐test. HT29 human xenografts were treated with anti‐PC1 (extracellular domain) inhibitory antibody and analyzed via immunohistochemistry to determine the in vivo role of PC1 in CRC. Clinical significance was assessed by examining PC1 and PC2 protein expression in CRC patients (immunohistochemistry). In vivo and clinical data were analyzed by non‐parametric tests, Kaplan‐Meier curves, log‐rank test and Cox model. All statistical tests were two‐sided. PC1 overexpression promotes epithelial‐to‐mesenchymal transition (EMT) in HCT116 cells, while PC2 overexpression results in upregulation of the mTOR pathway in SW480 cells. PC1 inhibition causes reduced cell proliferation in CRC cells inducing tumor necrosis and suppressing EMT in HT29 tumor xenografts. In clinical study, PC1 and PC2 overexpression associates with adverse pathological parameters, including invasiveness and mucinous carcinomas. Moreover, PC1 overexpression appears as an independent prognostic factor of reduced recurrence‐free survival (HR = 1.016, p = 0.03) and lowers overall survival probability, while aberrant PC2 expression predicts poor overall survival (p = 0.0468). These results support, for the first time, a direct link between mechanosensing polycystins (PC1 and PC2) and CRC progression.     

Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer‐initiating cells

The aim of the present study was to establish cancer stem‐like cell/cancer‐initiating cell (CSC/CIC)‐targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non‐CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8‐specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8‐derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non‐CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC‐targeting immunotherapy.     

Mesenchymal stromal cells induce epithelial‐to‐mesenchymal transition in human colorectal cancer cells through the expression of surface‐bound TGF‐β

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor‐associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow‐derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial‐to‐mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT‐related genes, downregulation of E‐cadherin and acquisition of mesenchymal morphology. These effects required cell‐to‐cell contact and were mediated by surface‐bound TGF‐β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC‐conditioned CRC cells were larger and characterized by higher vessel density, decreased E‐cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC‐conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT‐related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.     

53BP1 suppresses epithelial–mesenchymal transition by downregulating ZEB1 through microRNA‐200b/429 in breast cancer

Epithelial–mesenchymal transition (EMT) is an important mechanism of cancer invasion and metastasis. Although p53 binding protein 1 (53BP1) has been implicated in several biological processes, its function in EMT of human cancers has not yet been reported. Here, we show that 53BP1 negatively regulated EMT by modulating ZEB1 through targeting microRNA (miR)‐200b and miR‐429. Furthermore, 53BP1 promoted ZEB1‐mediated upregulation of E‐cadherin and also inhibited the expressions of mesenchymal markers, leading to increased migration and invasion in MDA‐MB‐231 breast cancer cells. Consistently, in MCF‐7 breast cancer cells, low 53BP1 expression reduced E‐cadherin expression, resulting in increased migration and invasion. These effects were reversed by miR‐200b and miR‐429 inhibition or overexpression. Sections of tumor xenograft model showed increased ZEB1 expression and decreased E‐cadherin expression with the downregulation of 53BP1. In 18 clinical tissue samples, expression of 53BP1 was positively correlated with miR‐200b and mir‐429 and negatively correlated with ZEB1. It was also found that 53BP1 was associated with lymph node metastasis. Taken together, these results suggest that 53BP1 functioned as a tumor suppressor gene by its novel negative control of EMT through regulating the expression of miR‐200b/429 and their target gene ZEB1.     

Combined targeting of high‐mobility group box‐1 and interleukin‐8 to control micrometastasis potential in gastric cancer

Micrometastasis is the major cause of treatment failure in gastric cancer (GC). Because epithelial‐to‐mesenchymal transition (EMT) is considered to develop prior to macroscopic metastasis, EMT‐promoting factors may affect micrometastasis. This study aimed to evaluate the role of extracellular high‐mobility group box‐1 (HMGB1) in EMT and the treatment effect of combined targeting of HMGB1 and interleukin‐8 (IL‐8) at early‐stage GC progression through interrupting EMT promotion. Extracellular HMGB1 was induced by human recombinant HMGB1 and pCMV‐SPORT6‐HMGB1 plasmid transfection. EMT activation was evaluated by immunoblotting, immunofluorescence and immunohistochemistry. Increased migration/invasion activities were evaluated by in vitro transwell migration/invasion assay using all histological types of human GC cell lines (N87, MKN28 SNU‐1 and KATOIII), N87‐xenograft BALB/c nude mice and human paired serum‐tissue GC samples. HMGB1‐induced soluble factors were measured by chemiluminescent immunoassay. Inhibition effects of tumor growth and EMT activation by combined targeting of HMGB1 and IL‐8 were evaluated in N87‐xenograft nude mice. Serum HMGB1 increases along the GC carcinogenesis and reaches maximum before macroscopic metastasis. Overexpressed extracellular HMGB1 promoted EMT activation and increased cell motility/invasiveness through ligation to receptor for advanced glycation end products. HMGB1‐induced IL‐8 overexpression contributed the HMGB1‐induced EMT in GC in vitro and in vivo. Blocking HMGB1 caused significant reduction of tumor growth, and addition of human recombinant IL‐8 rescues this antitumor effects. Our results imply the role of HMGB1 in EMT through IL‐8 mediation, and a potential mechanism of GC micrometastasis. Our observations suggest combination strategy of HMGB1 and IL‐8 as a promising diagnostic and therapeutic target to control GC micrometastasis.     

Side population of pancreatic cancer cells predominates in TGF‐β‐mediated epithelial to mesenchymal transition and invasion

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up‐regulated E‐cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF‐β, SP cells changed their shape into mesenchymal‐like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E‐cadherin expression level. TGF‐β induced EMT‐associated gene alteration such as reduction of E‐cadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)‐2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF‐β treatment, whereas MP cells did not respond to TGF‐β‐mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro‐invasion, and in vivo metastasis, as compared to MP cells. Because micro‐invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression. © 2008 UICC     

Multiple effects of the special AT‐rich binding protein 1 (SATB1) in colon carcinoma

SATB1 (special AT‐rich binding protein 1) is a global chromatin organizer regulating the expression of a large number of genes. Overexpression has been found in various solid tumors and positively correlated with prognostic and clinicopathological properties. In colorectal cancer (CRC), SATB1 overexpression and its correlation with poor differentiation, invasive depth, TNM (tumor, nodes, metastases) stage and prognosis have been demonstrated. However, more detailed studies on the SATB1 functions in CRC are warranted. In this article, we comprehensively analyze the cellular and molecular role of SATB1 in CRC cell lines with different SATB1 expression levels by using RNAi‐mediated knockdown. Using siRNAs with different knockdown efficacies, we demonstrate antiproliferative, cell cycle‐inhibitory and proapoptotic effects of SATB1 knockdown in a SATB1 gene dose‐dependent manner. Tumor growth inhibition is confirmed in vivo in a subcutaneous tumor xenograft mouse model using stable knockdown cells. The in‐depth analysis of cellular effects reveals increased activities of caspases‐3, ‐7, ‐8, ‐9 and other mediators of apoptotic pathways. Similarly, the analysis of E‐ and N‐cadherin, slug, twist, β‐catenin and MMP7 indicates SATB1 effects on epithelial–mesenchymal transition (EMT) and matrix breakdown. Our results also establish SATB1 effects on receptor tyrosine kinases and (proto‐)oncogenes such as HER receptors and Pim‐1. Taken together, this suggests a more complex molecular interplay between tumor‐promoting and possible inhibitory effects in CRC by affecting multiple pathways and molecules involved in proliferation, cell cycle, EMT, invasion and cell survival.     

Hypoxia promotes metastasis in human gastric cancer by up‐regulating the 67‐kDa laminin receptor

It has been reported that the 67‐kDa laminin receptor (67LR) is implicated in cancer metastasis. We recently showed that 37LRP, the 67LR precursor, is a hypoxia‐inducible factor 1 (HIF‐1) target gene exposed to hypoxia in gastric cancer. Here, we investigated the role of 67LR in hypoxic metastasis and invasion in gastric cancer. Immunohistochemical analysis, western blotting, and RT‐PCR assays revealed that 67LR was highly expressed in metastatic gastric cancers in vivo. Knockdown of the 67LR protein by RNA interference significantly decreased the adhesive, invasive, and in vivo metastatic abilities of the gastric cancer cell lines SGC7901 and MKN‐45. Western blot analysis showed that 67LR increased the expression of urokinase‐type plasminogen activator (uPA) and matrix metalloproteinase (MMP)‐9, and decreased tissue inhibitor of matrix metalloproteinase (TIMP)‐1 protein. We further showed that hypoxia induced 67LR expression in a time‐dependent manner and this induction was inhibited by HIF‐1 small‐interfering (si) RNA. Both ERK and JNK inhibitors significantly inhibited hypoxia‐induced expression of 67LR and the subsequent expression of uPA and MMP 9. SiRNA against 67LR or antibody against MMP9 and uPA significantly inhibited hypoxia‐induced in vitro invasive ability. Taken together, these results reveal that 67LR promotes the invasive and metastatic ability of the gastric cancer cells through increasing uPA and MMP 9 expression, with involvement of the ERK and JNK signal pathway in hypoxia‐induced 67 LR expressions and subsequent uPA and MMP9 expression. (Cancer Sci 2010)     

ATP‐P2Y2‐β‐catenin axis promotes cell invasion in breast cancer cells

Extracellular adenosine 5′‐triphosphate (ATP), secreted by living cancer cells or released by necrotic tumor cells, plays an important role in tumor invasion and metastasis. Our previous study demonstrated that ATP treatment in vitro could promote invasion in human prostate cancer cells via P2Y2, a preferred receptor for ATP, by enhancing EMT process. However, the pro‐invasion mechanisms of ATP and P2Y2 are still poorly studied in breast cancer. In this study, we found that P2Y2 was highly expressed in breast cancer cells and associated with human breast cancer metastasis. ATP could promote the in vitro invasion of breast cancer cells and enhance the expression of β‐catenin as well as its downstream target genes CD44, c‐Myc and cyclin D1, while P2Y2 knockdown attenuated above ATP‐driven events in vitro and in vivo. Furthermore, iCRT14, a β‐catenin/TCF complex inhibitor, could also suppress ATP‐driven migration and invasion in vitro. These results suggest that ATP promoted breast cancer cell invasion via P2Y2‐β‐catenin axis. Thus blockade of the ATP‐P2Y2‐β‐catenin axis could suppress the invasive and metastatic potential of breast cancer cells and may serve as potential targets for therapeutic interventions of breast cancer.     

Intratumoral macrophage counts correlate with tumor progression in colorectal cancer

Background

The role of intratumoral tumor‐associated macrophages (TAMs) in colorectal cancer (CRC) is not clear. We aim to examine the relationships of TAMs and the clinicopathologic features of CRC and the expression of matrix metalloproteinases (MMP)‐2 and MMP‐9.

Methods

Immunohistochemical staining of CD68, MMP‐2, and MMP‐9 was determined in tissue samples from CRC patients. To test the biological effect of macrophages on tumor cells, cancer cells were cocultured with macrophages and function change of cancer cells were examined.

Results

Intratumoral TAM count correlated with depth of invasion (P = 0.048), lymph node metastasis (P < 0.0001), and staging (P < 0.0001) of CRC. MMP‐2 and MMP‐9 expression was significantly associated with lymph node metastasis and staging. A significant association between intratumoral TAM counts and MMP‐2 (P < 0.0001) and MMP‐9 (P < 0.0001) expression was noted. When cocultured with macrophages, cancer cells increased their invasiveness and migration and elevated MMP‐2 and MMP‐9 secretion.

Conclusions

Intratumoral TAMs cause cancer cells to have a more aggressive behavior, and this may be due to an upregulation of tumor cell‐derived MMP‐2 and MMP‐9. Examination of intratumoral TAMs can serve as a progressive marker for CRC patients. J. Surg. Oncol. 2010;102:242–248. © 2010 Wiley‐Liss, Inc.     

Regulation of UHRF1 by microRNA‐9 modulates colorectal cancer cell proliferation and apoptosis

The UHRF1 protein is pivotal for DNA methylation and heterochromatin formation, leading to decreased expressions of tumor suppressor genes and contributing to tumorigenesis. However, the factors that modulate UHRF1 expression in colorectal cancer (CRC) remain unclear. Here we showed that, compared with corresponding normal tissues, UHRF1 was upregulated and microRNA‐9 (miR‐9) was downregulated in CRC tissues. The expression of UHRF1 was inversely correlated with overall survival rates of patients with CRC. Overexpression of miR‐9 in CRC cell lines significantly attenuated CRC cell proliferation and promoted cell apoptosis. The expression of UHRF1 was markedly reduced in pre‐miR‐9 transfected CRC cells. Using luciferase reporter assay, we confirmed that miR‐9 was a direct upstream regulator of UHRF1. Finally, analysis of miR‐9 and UHRF1 levels in human CRC tissues revealed that expression of miR‐9 was inversely correlated with UHRF1 expression. Collectively, our results offer in vitro validation of the concept that miR‐9 could repress the expression of UHRF1, and function as a tumor‐suppressive microRNA in CRC. It may serve as a prognostic and therapeutic marker for CRC.     

The MTHFR C677T and ΔDNMT3B C‐149T polymorphisms confer different risks for right‐ and left‐sided colorectal cancer

Etiological risk factors for proximal (right‐sided) colon cancers may be different to those of distal colon and rectal (left‐sided) cancers if these tumors develop along distinct pathways. The CpG Island Methylator Phenotype (CIMP+) occurs in approximately 15% of colorectal cancers (CRC) and predominantly in the proximal colon. CIMP+ tumors have frequent methylation of gene promoter regions and increased tissue folate levels. The aim here was to determine whether polymorphisms in 2 genes involved in cellular methyl group metabolism were associated with different risks for right‐ and left‐sided CRC. This population‐based case–control study involved 859 incident cases of CRC and 973 sex and age‐matched controls. Information on dietary folate and alcohol intake was obtained from food frequency questionnaires and information on the anatomical site of tumors from pathology reports. DNA was collected using FTA cards and genotyping performed for the MTHFR C677T and ΔDNMT3B C‐149T polymorphisms. The MTHFR 677 T allele was associated with increased risk for proximal colon cancer (adjusted odds ratio, AOR = 1.29) but decreased risk for distal cancers (AOR = 0.87). The increased risk for proximal cancers was especially pronounced in older individuals (AOR = 1.49) and those with a low folate diet (AOR = 1.67) or high alcohol consumption (AOR = 1.90). The ΔDNMT3B‐149 TT genotype was protective against proximal colon cancers (AOR = 0.65), but showed no association with the risk of distal colon and rectal cancers (AOR = 1.02). Epidemiological studies on dietary and genetic risk factors for CRC should take into account these may confer different risks for right‐ and left‐sided tumors. © 2009 UICC