Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Recent research has indicated that CXCR3/chemokines interactions that orchestrate haematopoetic cell movement are implicated in the metastatic process of malignant tumours, including that of CRC cells to lymph nodes. To date, however, the contribution of CXCR3 to liver and lung metastasis in CRC has not been addressed. To determine whether CXCR3 receptors regulate malignancy-related properties of CRC cells, we have used CXCR3-expressing CRC cell lines of human (HT29 cells) and murine (C26 cells) origins that enable the development of liver and lung metastases when injected into immunodeficient and immunocompetent mice, respectively, and assessed the effect of CXCR3 blockade using AMG487, a small molecular weight antagonist. In vitro, activation of CXCR3 on human and mouse CRC cells by its cognate ligands induced migratory and growth responses, both activities being abrogated by AMG487. In vivo, systemic CXCR3 antagonism by preventive or curative treatments with AMG487 markedly inhibited the implantation and the growth of human and mouse CRC cells within lung without affecting that in the liver. In addition, we measured increased levels of CXCR3 and ligands expression within lung nodules compared with liver tumours. Altogether, our findings indicate that activation of CXCR3 receptors by its cognate ligands facilitates the implantation and the progression of CRC cells within lung tissues and that inhibition of this axis decreases pulmonary metastasis of CRC in two murine tumour models.
chemokine receptor; metastasis; colon cancer; anti-tumour strategy; animal model
Although stromal cell-derived factor (SDF)-1α and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1α and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1α and CXCR4. The relationships between clinicopathological features and SDF-1α or CXCR4 expression were then analysed. Stromal cell-derived factor-1α and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1α and nuclear CXCR4 predicts LN metastasis in CRCs.
chemokines; SDF-1α; CXCR4; colorectal cancer; lymph node metastasis
Recent gene expression profiling analyses and gain- and loss-of-function studies performed with distinct prostate cancer (PC) cell models indicated that the alterations in specific gene products and molecular pathways often occur in PC stem/progenitor cells and their progenies during prostate carcinogenesis and metastases at distant sites, including bones. Particularly, the sustained activation of epidermal growth factor receptor (EGFR), hedgehog, Wnt/β-catenin, Notch, hyaluronan (HA)/CD44 and stromal cell–derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) during the epithelial-mesenchymal transition (EMT) process may provide critical functions for PC progression to locally invasive, metastatic and androgen-independent disease states and treatment resistance. Moreover, an enhanced glycolytic metabolism in PC stem/progenitor cells and their progenies concomitant with the changes in their local microenvironment, including the induction of tumor hypoxia and release of diverse soluble factors by tumor myofibroblasts, also may promote the tumor growth, angiogenesis and metastases. More particularly, these molecular transforming events may cooperate to upregulate Akt, nuclear factor (NF)-κB, hypoxia-inducible factors (HIFs) and stemness gene products such as Oct3/4, Sox2, Nanog and Bmi-1 in PC cells that contribute to their acquisition of high self-renewal, tumorigenic and invasive capacities and survival advantages during PC progression. Consequently, the molecular targeting of these deregulated gene products in the PC- and metastasis-initiating cells and their progenies represent new promising therapeutic strategies of great clinical interest for eradicating the total PC cell mass and improving current antihormonal treatments and docetaxel-based chemotherapies, thereby preventing disease relapse and the death of PC patients.
The canonical Wnt signalling pathway is activated in most sporadic colorectal cancers (CRCs). We previously reported that FZD7 functions as a receptor for the canonical Wnt signalling pathway in colon cancer cells.
Methods and results:
In this study, we examined the function of FZD7 in survival, invasion and metastatic capabilities of colon cancer cells. FZD7_siRNA transfection decreased cell viability of HT-29 and HCT-116 colon cancer cells. Expression of c-Jun, phosphorylation of JNK and c-Jun, and activation of RhoA were suppressed after FZD7_siRNA transfection into HCT-116 cells. In vitro invasion activity and Wnt target gene expression were also reduced in HCT-116 cells transfected with FZD7_siRNA. Liver metastasis of stable FZD7_siRNA HCT-116 cell transfectants in scid mice was decreased to 40–50% compared to controls. The mRNA levels of FZD7 in 135 primary CRC tissues were examined by real-time PCR. FZD7 mRNA levels were significantly higher in stage II, III or IV tumours than in non-tumour tissues (P<0.005), and overall survival was shorter in those patients with higher FZD7 expression (P<0.001).
These data suggest that FZD7 may be involved in enhancement of survival, invasion and metastatic capabilities of colon cancer cells through non-canonical Wnt signalling pathways as well as the canonical pathway.
frizzled-7; siRNA; colorectal cancer; metastasis; RhoA
It has been proposed in human colorectal cancers (CRC), a minority subset of cancer cells within tumors able to initiate tumor growth, defined as cancer stem cells (CSC). Solid human primary colonic and its ovarian metastatic cancer tissues were collected from fresh surgical samples and subsequent xenografts were established in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The resulting tumors were disaggregated into single-cell suspensions and a CD133 negative cell line (NANK) was newly established and analyzed by flow cytometry. Surface markers of progenitor cells were immunophenotypically analyzed, and expression of stem cell and cancer-related genes were characterized. Secreted angiogenesis-associated molecules were investigated by proteomic array technology. Finally, different number of NANK was implanted and their tumor-initiating properties were investigated in NOD/SCID mice. Intraperitoneal injection of NANK in NOD/SCID mice induced tumors with developing progressive peritoneal dissemination and ascites. NANK cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Noticeably, NANK lacked the expression of conventional CSC markers CD133 and CD44, self-renewal genes Oct-4 and Nanog, but showed the expression of an important gastrointestinal development marker CDX-2 and BMI-1 that is essential in regulating the proliferative activity of normal and leukemic stem cells. In addition, NANK secreted high amounts of important angiogeneic cytokines.
These results provide a novel and extensive model in human CSC for studying the generation and maintenance of phenotypic heterogeneity in CRC.
Cell line; cancer; metastasis; intraperitoneal dissemination; gene expression; cytokines; angiogenesis
In epithelial tumors, the platelet-derived growth factor receptor B (PDGFRB) is mainly expressed by stromal cells of mesenchymal origin. Tumor cells may also acquire PDGFRB expression following epithelial-to-mesenchymal transition (EMT), which occurs during metastasis formation. Little is known about PDGFRB signaling in colorectal tumor cells. We studied the relationship between PDGFRB expression, EMT, and metastasis in human colorectal cancer (CRC) cohorts by analysis of gene expression profiles. PDGFRB expression in primary CRC was correlated with short disease-free and overall survival. PDGFRB was co-expressed with genes involved in platelet activation, transforming growth factor beta (TGFB) signaling, and EMT in three CRC cohorts. PDGFRB was expressed in mesenchymal-like tumor cell lines in vitro and stimulated invasion and liver metastasis formation in mice. Platelets, a major source of PDGF, preferentially bound to tumor cells in a non-activated state. Platelet activation caused robust PDGFRB tyrosine phosphorylation on tumor cells in vitro and in liver sinusoids in vivo. Platelets also release TGFB, which is a potent inducer of EMT. Inhibition of TGFB signaling in tumor cells caused partial reversion of the mesenchymal phenotype and strongly reduced PDGFRB expression and PDGF-stimulated tumor cell invasion. These results suggest that PDGFRB may contribute to the aggressive phenotype of colorectal tumors with mesenchymal properties, most likely downstream of platelet activation and TGFB signaling.
The CXC chemokine ligand 12 (CXCL12)/stromal cell-derived factor-1 (SDF-1) and CXC receptor 4 (CXCR4) axis is involved in human colorectal cancer (CRC) carcinogenesis and can promote the progression of CRC. Interaction between CRC cells and endothelium is a key event in tumor progression. The aim of this study was to investigate the effect of SDF-1 on the adhesion of CRC cells.
Human CRC DLD-1 cells were used to study the effect of SDF-1 on intercellular adhesion molecule-1 (ICAM-1) expression and cell adhesion to endothelium.
SDF-1 treatment induced adhesion of DLD-1 cells to the endothelium and increased the expression level of the ICAM-1. Inhibition of ICAM-1 by small interfering RNA (siRNA) and neutralizing antibody inhibited SDF-1-induced cell adhesion. By using specific inhibitors and short hairpin RNA (shRNA), we demonstrated that the activation of ERK, JNK and p38 pathways is critical for SDF-1-induced ICAM-1 expression and cell adhesion. Promoter activity and transcription factor ELISA assays showed that SDF-1 increased Sp1-, C/EBP-β- and NF-κB-DNA binding activities in DLD-1 cells. Inhibition of Sp1, C/EBP-β and NF-κB activations by specific siRNA blocked the SDF-1-induced ICAM-1 promoter activity and expression. The effect of SDF-1 on cell adhesion was mediated by the CXCR4.
Our findings support the hypothesis that ICAM-1 up-regulation stimulated by SDF-1 may play an active role in CRC cell adhesion.
Colorectal cancer; Stromal cell-derived factor-1; Intercellular adhesion molecule-1; Cell adhesion; Transcriptional regulation
AIM: To evaluate the effect of nigericin on colorectal cancer and to explore its possible mechanism.
METHODS: The human colorectal cancer (CRC) cell lines HT29 and SW480 were treated with nigericin or oxaliplatin under the conditions specified. Cell viability assay and invasion and metastasis assay were performed to evaluate the effect of nigericin on CRC cells. Sphere-forming assay and soft agar colony-forming assay were implemented to assess the action of nigericin on the cancer stem cell properties of CRC cells undergone epithelial-mesenchymal transition (EMT).
RESULTS: Compared with oxaliplatin, nigericin showed more toxicity for the HT29 cell line (IC50, 12.92 ± 0.25 μmol vs 37.68 ± 0.34 μmol). A similar result was also obtained with the SW116 cell line (IC50, 15.86 ± 0.18 μmol vs 41.02 ± 0.23 μmol). A Boyden chamber assay indicated that a significant decrease in the number of HT29 cells migrating through polyvinylidene fluoride membrane was observed in the nigericin-treated group, relative to the vehicle-treated group [11 ± 2 cells per high-power field (HPF) vs 19.33 ± 1.52 cells per HPF, P < 0.05]. Compared to the control group, the numbers of HT29 cells invading through the Matrigel-coated membrane also decreased in the nigericin-treated group (6.66 ± 1.52 cells per HPF vs 14.66 ± 1.52 cells per HPF, P < 0.05). Nigericin also reduced the proportion of CD133+ cells from 83.57% to 63.93%, relative to the control group (P < 0.05). Nigericin decreased the number of spheres relative to the control group (0.14 ± 0.01 vs 0.35 ± 0.01, P < 0.05), while oxaliplatin increased the number of spheres relative to the control group (0.75 ± 0.02 vs 0.35 ± 0.01; P < 0.05). Nigericin also showed a decreased ability to form colonies under anchorage-independent conditions in a standard soft agar assay after 14 d in culture, relative to the control group (1.66 ± 0.57 vs 7 ± 1.15, P < 0.05), whereas the colony numbers were higher in the oxaliplatin group relative to the vehicle-treated controls (14.33 ± 0.57 vs 7 ± 1.15, P < 0.05). We further detected the expression of E-cadherin and vimentin in cells treated with nigericin and oxaliplatin. The results showed that HT29 cells treated with nigericin induced an increase in E-cadherin expression and a decrease in the vimentin expression relative to vehicle controls. In contrast, oxaliplatin downregulated the expression of E-cadherin and upregulated the expression of vimentin in HT29 cells relative to vehicle controls.
CONCLUSION: This study demonstrated that nigericin could partly reverse the EMT process during cell invasion and metastasis.
Colorectal cancer; Nigericin; Cancer invasion; Metastasis; Epithelial-mesenchymal transition; CD133; E-cadherin; Vimentin
The metastatic spread of tumor cells is one of the most common causes of mortality in cancer patients. The elucidation of the molecular mechanisms that underlie the formation of metastatic colonies has been one of the major objectives of cancer research. Organ-specific colonization of cancer cells is a significant and noteworthy feature of metastasis. Colorectal cancer (CRC) is one of the most common causes of cancer-related mortality. The liver is commonly the sole site of metastasis for CRC and represents a major cause of mortality in CRC patients. However, what regulates CRC cell metastasis into liver and the reasons for the liver-specific metastasis of CRC have yet to be adequately elucidated. Recent progress provides indications and a conceptual framework with which to investigate this issue. This review evaluated experimental and clinical evidence to support a mechanistic role for circulation patterns and microvessels in liver, metastasis-related genes, chemokines and their receptors, and cellular adhesion molecules in the process of CRC liver metastasis.
colorectal cancer; liver metastasis; organ selectivity
Stem cells are thought to contribute to tissue regeneration as well as carcinogenesis. Ulcerative colitis-associated colorectal cancer (UC-CRC) has shown distinct characteristics compared with those of sporadic CRC. The aim of this study was to evaluate the expression of stem cell markers CD133, OCT4 and NANOG in UC-CRC and the inflamed colonic epithelium of UC patients. Total RNAs of UC-CRC (n=6), inflamed colonic epithelium (n=24), sporadic CRC (n=37) and adjacent normal colonic epithelium (n=37) were isolated from formalin-fixed, paraffin-embedded specimens using microdissection techniques in order to purify colonic epithelial cells. Relative mRNA levels of CD133 (PROM), OCT4 (POU5F1) and NANOG were measured using real-time reverse transcription polymerase chain reaction. Three stem cell markers were also investigated immunohistochemically. PROM, POU5F1 and NANOG levels were found to be significantly lower in UC-CRC than in inflamed colonic epithelium of UC patients. By contrast, sporadic CRC showed a significantly higher expression of PROM, POU5F1 and NANOG compared with adjacent normal colonic epithelium. POU5F1 and NANOG levels were significantly lower in UC-CRC than in sporadic CRC. PROM and NANOG levels in inflamed colonic epithelium were significantly higher among younger UC patients (P<0.05). Longer disease duration was significantly associated with lower PROM expression (P=0.0117). No significant difference was found in PROM levels between UC-CRC and inflamed colonic epithelium in patients with longer disease duration. UC-CRC showed different expression profiles of stem cell markers compared with sporadic CRC. Decreases in PROM expression of inflamed colonic epithelium may identify UC patients at high risk for the development of UC-CRC.
CD133; OCT4; NANOG; ulcerative colitis-associated colorectal cancer; ulcerative colitis
CXCR4, the receptor for the chemokine stromal-derived factor 1 (SDF-1), has been shown to mediate many of the processes essential for cancer progression such as tumor cell proliferation, metastasis, and angiogenesis. To understand the role of CXCR4 in the biology of neuroblastoma, a disease that presents with wide spread metastases in over 50% of patients, we screened ten patient derived-neuroblastoma cell-lines for basal CXCR4 expression and sought to identify characteristics that correlate with tumor cell phenotype.
All cell lines expressed CXCR4 mRNA at variable levels, that correlated well with three distinct classes of CXCR4 surface expression (low, moderate, or high) as defined by flow cytometry. Analysis of the kinetics of CXCR4 surface expression on moderate and high expressing cell lines showed a time-dependent down-regulation of the receptor that directly correlated with cell confluency, and was independent of SDF1. Cell lysates showed the presence of multiple CXCR4 isoforms with three major species of approximately 87, 67 and 55 kDa associating with high surface expression, and two distinct species of 45 and 38 kDa correlating with low to null surface expression. Western blot analysis of CXCR4 immunoprecipitates showed that the 87 and 67 kDa forms were ubiquitinated, while the others were not. Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.
Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism. Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.
Precursors of the hormone gastrin, progastrin and glycine-extended gastrin (G-gly), have been detected in colorectal polyps and tumours, and in the blood of patients with colorectal cancer (CRC), while their expression is lower in healthy subjects. The surface glycoproteins CD133 and CD44 have been identified as possible markers for CRC stem cells. Our aims were to investigate whether progastrin and G-gly are expressed by CD133-positive cells in human CRC tissues and in the human CRC cell line DLD-1, and to determine whether this expression is biologically relevant. The great majority of the cells expressing CD133 also expressed gastrin precursors in both DLD-1 cells, which retain a stem cell-like subpopulation, and human CRC specimens. The CD133high/CD44high/progastrinhigh cells gave rise to larger tumours in SCID mice compared to CD133low/CD44low/progastrinlow cells. The CD133high/CD44high/progastrinhigh cells displayed enhanced activation of the signalling molecules JAK2, STAT3, ERK1/2 and Akt, known to regulate the induction of proliferation and/or survival by gastrin precursors. Moreover, downregulation of the gastrin gene in DLD-1 cells reduced the expression of cancer stem cell markers and abolished tumour development in SCID mice. We conclude that gastrin precursors may provide a target for therapies directed against the cells responsible for tumour development and recurrence.
CD133; cancer stem cell; tumour-initiating cell; colon; cancer; gastrin
JC Virus (JCV), a human polyomavirus, is frequently present in colorectal cancers (CRCs). JCV large T-Ag (T-Ag) expressed in approximately half of all CRC's, however, its functional role in CRC is poorly understood. We hypothesized that JCV T-Ag may mediate metastasis in CRC cells through increased migration and invasion.
Material and Methods
CRC cell lines (HCT116 and SW837) were stably transfected with JCV early transcript sequences cloned into pCR3 or empty vectors. Migration and invasion assays were performed using Boyden chambers. Global gene expression analysis was performed to identify genetic targets and pathways altered by T-Ag expression. Microarray results were validated by qRT-PCR, protein expression analyses and immunohistochemistry. Matching primary CRCs and liver metastases from 33 patients were analyzed for T-Ag expression by immunohistochemistry.
T-Ag expressing cell lines showed 2 to 3-fold increase in migration and invasion compared to controls. JCV T-Ag expression resulted in differential expression of several genetic targets, including genes that mediate cell migration and invasion. Pathway analysis suggested a significant involvement of these genes with AKT and MAPK signaling. Treatment with selective PI3K/AKT and MAPK pathway inhibitors resulted in reduced migration and invasion. In support of our in-vitro results, immunohistochemical staining of the advanced stage tumors revealed frequent JCV T-Ag expression in metastatic primary tumors (92%) as well as in their matching liver metastasis (73%).
These data suggest that JCV T-Ag expression in CRC associates with a metastatic phenotype, which may partly be mediated through the AKT/MAPK signaling pathway. Frequent expression of JCV T-Ag in CRC liver metastasis provides further clues supporting a mechanistic role for JCV as a possible mediator of cellular motility and invasion in CRC.
Cancer metastasis is a major clinical problem that contributes to unsuccessful therapy. Augmenting evidence indicates that metastasizing cancer cells employ several mechanisms that are involved in developmental trafficking of normal stem cells. Stromal-derived factor-1 (SDF-1) is an important α-chemokine that binds to the G-protein-coupled seven-transmembrane span CXCR4. The SDF-1-CXCR4 axis regulates trafficking of normal and malignant cells. SDF-1 is an important chemoattractant for a variety of cells including hematopoietic stem/progenitor cells. For many years, it was believed that CXCR4 was the only receptor for SDF-1. However, several reports recently provided evidence that SDF-1 also binds to another seven-transmembrane span receptor called CXCR7, sharing this receptor with another chemokine family member called Interferon-inducible T-cell chemoattractant (I-TAC). Thus, with CXCR7 identified as a new receptor for SDF-1, the role of the SDF-1-CXCR4 axis in regulating several biological processes becomes more complex. Based on the available literature, this review addresses the biological significance of SDF-1’s interaction with CXCR7, which may act as a kind of decoy or signaling receptor depending on cell type. Augmenting evidence suggests that CXCR7 is involved in several aspects of tumorogenesis and could become an important target for new anti-metastatic and anti-cancer drugs.
SDF-1; CXCR7; CXCR4; cancer metastasis
Chronic inflammation-promoted metastasis has been considered as a major challenge in cancer therapy. Pro-inflammatory cytokine TNFα can induce cancer invasion and metastasis associated with epithelial–mesenchymal transition (EMT). However, the underlying mechanisms are not entirely clear. In this study, we showed that TNFα induces EMT in human HCT116 cells and thereby promotes colorectal cancer (CRC) invasion and metastasis. TNFα-induced EMT was characterized by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin and fibronectin with a concomitant decrease of E-cadherin and Zona occludin-1(ZO-1). TNFα treatment also increased the expression of transcription factor Snail, but not Slug, ZEB1 and Twist. Overexpression of Snail induced a switch from E-cadherin to N-cadherin expression in HCT116 cells, which is a characteristic of EMT. Conversely, knockdown of Snail significantly attenuated TNFα-induced EMT in HCT116 cells, suggesting that Snail plays a crucial role in TNFα-induced EMT. Interestingly, exposure to TNFα rapidly increased Snail protein expression and Snail nuclear localization but not mRNA level upregulation. Finally, we demonstrated that TNFα elevated Snail stability by activating AKT pathway and subsequently repressing GSK-3β activity and decreasing the association of Snail with GSK-3β. Knockdown of GSK-3β further verified our finding. Taken together, these results revealed that AKT/GSK-3β-mediated stabilization of Snail is required for TNFα-induced EMT in CRC cells. Our study provides a better understanding of inflammation-induced CRC metastasis.
AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells.
METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining.
RESULTS: CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133hi and CD133- cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133- cells showed that CD133 was highly expressed in colorectal cancer cell lines.
CONCLUSION: Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.
CD133; Colon cancer cells; Tumorigenesis; Cancer stem cells
Chemotactic cytokines (chemokines) can help regulate tumor cell invasion and metastasis. Here, we show that chemokine 25 (CCL25) and its cognate receptor chemokine receptor 9 (CCR9) inhibit colorectal cancer (CRC) invasion and metastasis. We found that CCR9 protein expression levels were highest in colon adenomas and progressively decreased in invasive and metastatic CRCs. CCR9 was expressed in both primary tumor cell cultures and colon-cancer-initiating cell (CCIC) lines derived from early-stage CRCs but not from metastatic CRC. CCL25 stimulated cell proliferation by activating AKT signaling. In vivo, systemically injected CCR9+ early-stage CCICs led to the formation of orthotopic gastrointestinal xenograft tumors. Blocking CCR9 signaling inhibited CRC tumor formation in the native gastrointestinal CCL25+ microenvironment, while increasing extraintestinal tumor incidence. NOTCH signaling, which promotes CRC metastasis, increased extraintestinal tumor frequency by stimulating CCR9 proteasomal degradation. Overall, these data indicate that CCL25 and CCR9 regulate CRC progression and invasion and further demonstrate an appropriate in vivo experimental system to study CRC progression in the native colon microenvironment.
Renal cell carcinoma (RCC) is characterized by organ-specific metastases. The chemokine stromal derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 have been suggested to regulate organ-specific metastasis in various other cancers. On this basis, we hypothesized that the biological axis of CXCL12 via interaction with its receptor, CXCR4, is a major mechanism for RCC metastasis. We demonstrated that CXCR4 was significantly expressed on circulating cytokeratin+ RCC cells from patients with known metastatic RCC. We detected up-regulation of CXCR4 mRNA and protein levels on a human RCC cell line by either knockdown of the von Hippel-Lindau (VHL) tumor suppressor protein, or incubating the cells under hypoxic conditions. The enhanced CXCR4 expression was mediated through the interaction of the Hypoxia Inducible Factor-1α (HIF-1α) with the promoter region of the CXCR4 gene. Furthermore, the expression of CXCR4 on human RCC directly correlated with their metastatic ability in vivo in both heterotopic and orthotopic SCID mouse models of human RCC. Neutralization of CXCL12 in SCID mice abrogated metastasis of RCC to target organs expressing high levels of CXCL12; without altering tumor cell proliferation, apoptosis, or tumor-associated angiogenesis. Therefore, our data suggest that the CXCL12/CXCR4 biological axis plays an important role in regulating the organ-specific metastasis of RCC.
Introduction: Over 50% of patients with colorectal cancer (CRC) will progress and/or develop metastases. Biomarkers capable of predicting progression, risk stratification and therapeutic benefit are needed. Cancer stem cells are thought to be responsible for tumor initiation, dissemination and treatment failure. Therefore, we hypothesized that CRC cancer stem cell markers (CRCSC) will identify a group of patients at high risk for progression.
Methods: Paraffin-embedded tissue cores of normal (n=8), and histopathologically well-defined primary (n= 30) and metastatic (n=10) CRC were arrayed in duplicate on tissue microarrays (TMAs). Expression profiles of non-CD133 CRCSC (CD29, CD44, ALDH1A1, ALDH1B1, EpCam, and CD166) were detected by immunohistochemistry and the association with clinicopathological data and patient outcomes was determined using standard statistical methodology. An independent pathologist, blinded to the clinical data scored the samples. Scoring included percent positive cells (0 to 4, 0 = <10%, 1 = 10 - 24%, 2 = 25 - 49%, 3 = 50 - 74%, 4 = 75 - 100%), and the intensity of positively stained cells (0 to 4; 0 = no staining, 1 = diminutive intensity, 2 = low intensity, 3 = intermediate intensity, 4 = high intensity). The pathologic score represents the sum of these two values, reported in this paper as a combined IHC staining score (CSS).
Results: Of 30 patients 7 were AJCC stage IIA, 10 stage IIIB, 7 stage IIIC and 6 stage IV. Median follow-up was 113 months. DFI was 17 months. Median overall survival (OS) was not reached. Stage-specific OS was: II - not reached; III - not reached; IV - 11 months. In a univariate analysis, poor OS was associated with loss of CD29 expression; median OS, 32 months vs. not reached for CSS 3-7 vs. >7.5, respectively; p=0.052 comparing entire curves, after adjustment. In a Cox model analysis, loss of CD29 exhibited a trend toward association with survival (p=0.098) after adjusting for the effect of stage (p=0.0076). Greater expression of ALDH1A1 was associated with increasing stage (p=0.042 over stages 2, 3b, 3c, and 4) while loss of CD29 expression exhibited a trend toward being associated with stages 3 and 4 (p=0.08). Compared to normal colon tissue, primary tumors were associated with increased expression of ALDH1B1 (p=0.008). ALD1H1B1 expression level differed according to whether the tumor was moderately or poorly differentiated, well differentiated, or mucinous; the highest expression levels were associated with moderately or poorly differentiated tumors (p=0.011). Lymph node metastases were associated with a trend toward decreased expression of EpCAM (p = 0.06) when comparing 0 vs. 1 vs. 2+ positive lymph nodes, as was CD29 (p = 0.08) when comparing 0 vs. any positive lymph nodes. Compared to normal colon tissue metastatic colon cancers from different patients were associated with increased ALDH1B1 expression (p=0.001) whereas CD29 expression was higher in normal colonic tissue (p=0.014).
Conclusion: CD29 may be associated with survival as well as clinical stage and number of lymph nodes. ALDH1B1 expression was associated with differentiation as well as type of tissue evaluated. ALDH1A1 was associated with clinical stage, and decreased EpCAM expression was found in patients with advanced lymph node stage. CRCSCs may be useful biomarkers to risk stratify, and estimate outcomes in CRC. Larger prospective studies are required to validate the current findings.
colon cancer; staging; lymph node; cancer recurrence; overall survival; prognosis; biomarkers; cancer stem cells.
Malignant progression in cancer requires populations of tumor-initiating cells
(TICs) endowed with unlimited self renewal, survival under stress, and
establishment of distant metastases. Additionally, the acquisition of invasive
properties driven by epithelial-mesenchymal transition (EMT) is critical for the
evolution of neoplastic cells into fully metastatic populations. Here, we
characterize 2 human cellular models derived from prostate and bladder cancer
cell lines to better understand the relationship between TIC and EMT programs in
local invasiveness and distant metastasis. The model tumor subpopulations that
expressed a strong epithelial gene program were enriched in highly metastatic
TICs, while a second subpopulation with stable mesenchymal traits was
impoverished in TICs. Constitutive overexpression of the transcription factor
Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene
program and suppressed their self renewal and metastatic phenotypes. Conversely,
knockdown of EMT factors in the mesenchymal-like prostate cancer cell
subpopulation caused a gain in epithelial features and properties of TICs. Both
tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like
prostate cancer subpopulation enhanced the in vitro invasiveness of the
metastatic epithelial subpopulation and, in vivo, promoted the escape of the
latter from primary implantation sites and accelerated their metastatic
colonization. Our models provide new insights into how dynamic interactions
among epithelial, self-renewal, and mesenchymal gene programs determine the
plasticity of epithelial TICs.
Liver metastasis is a major cause of mortality from colorectal cancer (CRC). However, mechanisms underlying this process are largely unknown. Osteopontin (OPN) is a secreted phosphorylated glycoprotein that is involved in tumor migration and metastasis. The role of OPN in cancer is currently unclear. In this study, OPN mRNA was examined in tissues from CRC, adjacent normal mucosa, and liver metastatic lesions using quantitative real-time PCR analysis. The protein expression of OPN and its receptors (integrin αv and CD44 v6) was detected by using an immunohistochemical (IHC) method. The role of OPN in liver metastasis was studied in established colon cancer Colo-205 and SW-480 cell lines transfected with sense- or antisense-OPN eukaryotic expression plasmids by flow cytometry and cell adhesion assay. Florescence redistribution after photobleaching (FRAP) was used to study gap functional intercellular communication (GJIC) among OPN-transfected cells. It was found that OPN was highly expressed in metastatic hepatic lesions from CRC compared to primary CRC tissue and adjacent normal mucosa. The expression of OPN mRNA in tumor tissues was significantly related with the CRC stages. OPN expression was also detected in normal hepatocytes surrounding CRC metastatic lesions. Two known receptors of OPN, integrin αv and CD44v6 proteins, were strongly expressed in hepatocytes from normal liver. CRC cells with forced OPN expression exhibited increased heterotypic adhesion with endothelial cells and weakened intercellular communication. OPN plays a significant role in CRC metastasis to liver through interaction with its receptors in hepatocytes, decreased homotypic adhesion, and enhanced heterotypic adhesion.
Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.
CD39; NTPDase1; P2 receptors; Colorectal cancer; MC-26 cancer cell line
Chemokines play an important role in cancer metastasis by modulating the directional cell movement and migration of tumor cells. The most commonly overexpressed chemokine receptor in human cancer is CXCR4. Once activated by its ligand CXCL12 (stromal cell-derived factor-1 ligand/SDF1), CXCR4 stimulates several key migratory, proliferative and survival signaling cellular pathways. CXCR4 is expressed in small-cell lung carcinoma (SCLC) cells and other tumors. To further characterize the role of chemokines in tumor-to-tumor metastasis, we analyzed the tissue expression of CXCR4 and CXCL12 in the surgical specimen of a patient with this phenomenon. We performed immunohistochemical analysis for the expression of CXCR4 and CXCL12 in metastatic tumor tissue of a 69-year-old Caucasian male with extensive SCLC metastatic to a renal oncocytoma. The oncocytoma tissue harboring SCLC showed CXCL12 expression, but not CXCR4. A high expression of the two molecules was found in a normal renal parenchymal control. Our results suggest that CXCR4 and CXCL12 plays a role in this condition, but their expression may be affected by the microenvironment of the harboring malignancy. Further characterization of these phenomena is needed to shed light on the biological mechanisms of tumor metastasis.
stromal cell-derived factor ligand 1 α; stromal cell-derived factor ligand 1 α receptor; small-cell lung carcinoma cells
Epithelial–mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail-overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC-like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC.
Cancer stem cells; colorectal cancer; EMT; migration; Snail
We have demonstrated that the α-chemokine stromal-derived factor (SDF)-1-CXCR4 axis plays an important role in rhabdomyosarcoma (RMS) metastasis. With the recent descriptionof CXCR7, a new receptor for SDF-1 that also binds the interferon-inducible T-cell alpha chemoattractant (ITAC) chemokine, we became interested in the role of the CXCR7-SDF-1/ITAC axis in RMS progression. To address this issue, we evaluated 6 highly metastatic alveolar (A)RMS and 3 less metastatic embryonal (E)RMS cell lines and found that all these cell lines express CXCR7. While CXCR4 was expressed at a much higher level by highly metastatic ARMS lines, CXCR7 was present at a high level on ERMS lines. We also noticed that CXCR7 expression on RMS cells was downregulated in hypoxic conditions. More importantly, the CXCR7 receptor on RMS cell lines was functional after stimulation with ITAC and SDF-1 as evidenced by mitogen-activated protein kinase (MAPK)p42/44 and AKT phosphorylation as well as CXCR7 internalization, chemotaxis, cell motility, and adhesion assays. Similarly to CXCR4, signaling from activated CXCR7 was not associated with increased RMS proliferation or cell survival. Moreover, CXCR7+ RMS cells responded to SDF-1 and I-TAC in the presence of CXCR4 antagonists (T140, AMD3100). Furthermore, while intravenous injection of RMS cells with overexpressed CXCR7 resulted in increased seeding efficiency of tumor cells to bone marrow, CXCR7 downregulation showed the opposite effect. In conclusion, the CXCR7-SDF-1/ITAC axis is involved in the progression of RMS; targeting of the CXCR4-SDF-1 axis alone without simultaneous blockage of CXCR7 will be an inefficient strategy for inhibiting SDF-1-mediated pro-metastatic responses of RMS cells.
Rhabdomyosarcoma; SDF-1; I-TAC; CXCR4; CXCR7