Hematogenous Cancer Metastasis Research Articles

Endothelial CLEC-1b plays a protective role against cancer hematogenous metastasis

Metastasis, which is the spread of cancer cells into distant organs, is a critical determinant of prognosis in patients with cancer, and blood vessels are the major route for cancer cells to spread systemically. Extravasation is a critical process for the hematogenous metastasis; however, its underlying molecular mechanisms remain poorly understood. Here, we identified that senescent ECs highly express C-type lectin domain family 1 member B (CLEC-1b), and that endothelial CLEC-1b inhibits the hematogenous metastasis of a certain type of cancer. CLEC-1b expression was enhanced in ECs isolated from aged mice, senescent cultured human ECs, and ECs of aged human. CLEC-1b overexpression in ECs prevented the disruption of endothelial integrity, and inhibited the transendothelial migration of cancer cells expressing podoplanin (PDPN), a ligand for CLEC-1b. Notably, target activation of CLEC-1b in ECs decreased the hematogenous metastasis in the lungs by cancer cells expressing PDPN in mice. Our data reveal the protective role of endothelial CLEC-1b against cancer hematogenous metastasis. Considering the high CLEC-1b expression in senescent ECs, EC senescence may play a beneficial role with respect to the cancer hematogenous metastasis.

Regional lymph nodes and hematogenous metastasis of cancer

Aim. Morphological study of the microvasculature of regional lymph nodes in relation to the cancer of the lymph nodes as possible additional or alternative metastasis pathways. Methods. The lymph nodes of 150 cancer patients (1263 nodes in total), regional to cancer of various localization, were studied. Histological sections staining with hematoxylin and eosin by Van-Giesons method, pyronine by Brachets method, toluidine blue and picro-Mallory were prepared. An immunohistochemical study was performed using monoclonal antibodies to pan-cytokeratins, CD31, type IV collagen, CD3, CD20, and CD68. The area of metastases to the lymph nodes was determined by using a morphometric grid and used to identify the four study groups. In addition, the immunomorphological reactions of the lymph nodes were taken into account in each group. Results. It was identified that the microvasculature of the lymph nodes can be involved in the metastatic process along with the lymphatic pathways. At the same time, there is a decrease in vascular wall function and violation of the rheological properties of blood, accompanied by the deposition of intra- and extravascular fibrin. Hematogenous metastasis is largely influenced by the state of lymph node sinuses, in which blood is found, and in some observations by the expression of CD31 (a marker of blood endothelium). Hematogenous dissemination of cancer often begins after the appearance of lymph node metastases. The greater the anatomical extent of lymph node metastases, the more often tumor cells are present in the blood vessels. In addition, an isolated lesion of the microvasculature with the presence of tumor cells in the extranodal vessels without metastases in the lymph node itself was revealed. It was observed that the invasion of tumor cells into the microvasculature depended on the immunomorphological reactions of the lymph nodes. Conclusion. The microvasculature of regional lymph nodes can be both an additional and an alternative lymphogenous metastasis pathway of cancer; at the same time, vascular invasion is accompanied by microcirculation disorders and depends on the volume of metastases and the immunomorphological reactions of the lymph nodes.

Anti-metastatic effect of methylprednisolone targeting vascular endothelial cells under surgical stress

Perioperative systemic inflammation induced by surgical stress elevates the risk of hematogenous cancer metastasis. This study investigated the anti-metastatic effects and mechanisms of methylprednisolone (MP) administration for surgical stress. We examined the effects of MP on the expression of adhesion molecules in human vascular endothelial cells and in a murine hepatic metastasis model under lipopolysaccharide (LPS) administration, which mimics systemic inflammation induced by surgical stress. Serum E-selectin level was measured in blood samples obtained from 32 gastric cancer patients who were randomly assigned to treat preoperatively with or without MP. The expression of E-selectin in LPS-induced vascular endothelial cells was suppressed by MP. An adhesion assay showed the number of LPS-induced adherent tumour cells was significantly lower following MP. In the in vivo study, LPS significantly elevated the number of hepatic metastases, but pretreatment with MP before LPS significantly inhibited this elevation. The LPS-induced expression of E-selectin in the vascular endothelium of the portal vein was suppressed by MP. In human clinical samples, serum E-selectin level was significantly decreased by preoperative MP. Suppression of surgically induced systemic inflammation by MP administration might prevent hematogenous cancer metastases by suppressing the induction of E-selectin expression in the vascular endothelium.

Platelets and cancer-associated thrombosis: focusing on the platelet activation receptor CLEC-2 and podoplanin.

Patients with cancer have an increased risk of thromboembolism, which is the second leading cause of death in these patients. Several mechanisms of the prothrombotic state in these patients have been proposed. Among them are a platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2), and its endogenous ligand podoplanin, which are the focus of this review. CLEC-2 is almost specifically expressed in platelets/megakaryocytes in humans. A membrane protein, podoplanin is expressed in certain types of cancer cells, including squamous cell carcinoma, brain tumor, and osteosarcoma, in addition to several normal tissues, including kidney podocytes and lymphatic endothelial cells but not vascular endothelial cells. In the bloodstream, podoplanin induces platelet activation by binding to CLEC-2 and facilitates hematogenous cancer metastasis and cancer-associated thrombosis. In an experimental lung metastasis model, the pharmacological depletion of CLEC-2 from platelets in mice resulted in a marked reduction of lung metastasis of podoplanin-expressing B16F10 cells. Control mice with B16F10 orthotopically inoculated in the back skin showed massive thrombus formation in the lungs, but the cancer-associated thrombus formation in CLEC-2-depleted mice was significantly inhibited, suggesting that CLEC-2-podoplanin interaction stimulates cancer-associated thrombosis. Thromboinflammation induced ectopic podoplanin expression in vascular endothelial cells or macrophages, which may also contribute to cancer-associated thrombosis. CLEC-2 depletion in cancer-bearing mice resulted in not only reduced cancer-associated thrombosis but also reduced levels of plasma inflammatory cytokines, anemia, and sarcopenia, suggesting that cancer-associated thrombosis may cause thromboinflammation and cancer cachexia. Blocking CLEC-2-podoplanin interaction may be a novel therapeutic strategy in patients with podoplanin-expressing cancer.

In Vivo Flow Cytometry of Extremely Rare Circulating Cells

Circulating tumor cells (CTCs) are of great interest in cancer research, but methods for their enumeration remain far from optimal. We developed a new small animal research tool called “Diffuse in vivo Flow Cytometry” (DiFC) for detecting extremely rare fluorescently-labeled circulating cells directly in the bloodstream. The technique exploits near-infrared diffuse photons to detect and count cells flowing in large superficial arteries and veins without drawing blood samples. DiFC uses custom-designed, dual fiber optic probes that are placed in contact with the skin surface approximately above a major vascular bundle. In combination with a novel signal processing algorithm, DiFC allows counting of individual cells moving in arterial or venous directions, as well as measurement of their speed and depth. We show that DiFC allows sampling of the entire circulating blood volume of a mouse in under 10 minutes, while maintaining a false alarm rate of 0.014 per minute. In practice, this means that DiFC allows reliable detection of circulating cells below 1 cell per mL. Hence, the unique capabilities of DiFC are highly suited to biological applications involving very rare cell types such as the study of hematogenous cancer metastasis.

The expanding constellation of immune checkpoints: a DNAMic control by CD155.

The clinical benefits that have been achieved for a group of cancer patients with metastatic disease on checkpoint inhibitor therapy have kindled intense interest in understanding tumor-induced escape from T lymphocyte control. Other lymphoid cells also participate in tumor control; in particular, NK cells can limit hematogenous cancer metastasis spread and are also subject to negative regulation by developing cancers. In this issue of the JCI, Li and colleagues define an unanticipated role for the stress-induced protein CD155 in cancer metastasis. The presence of CD155 on the surface of cancer cells was shown to promote tumor invasiveness, while its upregulation in tumor environment-infiltrating myeloid cells restrained antitumor immunity by impairing antitumor T lymphocytes and NK cell function. Together, these results support further exploration of strategies for targeting CD155.

Angiotensin II promotes pulmonary metastasis of melanoma through the activation of adhesion molecules in vascular endothelial cells

Hypertension is considered as one of the cancer progressive factors, and often found comorbidity in cancer patients. Renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, and angiotensin II (Ang II) is well known pressor peptide associated with RAS. Ang II has been reported to accelerate progression and metastasis of cancer cells. However, its precise mechanisms have not been fully understood. In this study, we sought to elucidate the mechanisms by which Ang II exacerbates hematogenous metastasis in mouse melanoma cells, focusing the adhesion pathway in vascular endothelial cells. For this purpose, B16/F10 mouse melanoma cells, which do not express the Ang II type 1 receptor (AT1R), were intravenously injected into C57BL/6 mice. Two weeks after cell injection, the number of lung metastatic colonies was significantly higher in the Ang II-treated group (1 μg/kg/min) than in the vehicle-treated group. The AT1R blocker valsartan (40 mg/kg/day), but not the calcium channel blocker amlodipine (5 or 10 mg/kg/day), significantly suppressed the effect of Ang II. In endothelium-specific Agtr1a knockout mice, Ang II-mediated acceleration of lung metastases of melanoma cells was significantly diminished. Ang II treatment significantly increased E-selectin mRNA expression in vascular endothelial cells collected from lung tissues, and thus promoted adherence of melanoma cells to the vascular endothelium. Ang II-accelerated lung metastases of melanoma cells were also suppressed by treatment with anti-E-selectin antibody (20 mg/kg). Taken together, Ang II-treatment exacerbates hematogenous cancer metastasis by promoting E-selectin-mediated adhesion of cancer cells to vascular endothelial cells.

C-type lectins facilitate tumor metastasis.

Metastasis, a life-threatening complication of cancer, leads to the majority of cases of cancer-associated mortality. Unfortunately, the underlying molecular and cellular mechanisms of cancer metastasis remain to be fully elucidated. C-type lectins are a large group of proteins, which share structurally homologous carbohydrate-recognition domains (CRDs) and possess diverse physiological functions, including inflammation and antimicrobial immunity. Accumulating evidence has demonstrated the contribution of C-type lectins in different steps of the metastatic spread of cancer. Notably, a substantial proportion of C-type lectins, including selectins, mannose receptor (MR) and liver and lymph node sinusoidal endothelial cell C-type lectin, are important molecular targets for the formation of metastases in vitro and in vivo. The present review summarizes what has been found regarding C-type lectins in the lymphatic and hematogenous metastasis of cancer. An improved understanding the role of C-type lectins in cancer metastasis provides a comprehensive perspective for further clarifying the molecular mechanisms of cancer metastasis and supports the development of novel C-type lectins-based therapies the for prevention of metastasis in certain types of cancer.

Abstract P4-04-07: Tartrate-resistant Acid Phosphatase (TRAcP)-Expressed Tumor-Associated Macrophages Promote Breast Cancer Progression

Abstract Purpose: Tumor-associated macrophages (TAMs) can promote or dampen breast cancer progression, depending on phenotypic changes within the cancer microenvironment. Two contrasting activation states were described in the literature: the M1 anti-tumoral and M2 pro-tumoral subtype of TAMs. In clinical observation, we analyzed the TRAcP expression in breast cancer TAM and correlated to patient's chemotherapy responses and survival. In animal models, we aim to investigate the tumor-promoting or inhibiting properties among tartrate-resistant acid phosphatase (TRAcP) expressed TAMs. Methods: TRAcP expression, T lymphocytes, and other macrophage relevant markers were determined by immunohistochemical staining in the human breast cancer tissue from the patients before neoadjuvant chemotherapy. The expression of TRAcP within the TAMs were calculated and correlated to the patient's chemotherapy responsiveness and outcome. The polarized M1/M2 macrophages and breast cancer hematogenous metastasis were created in the animal model. Activated macrophages from TRAcP+/+ or TRAcP−/− mice were injected with JC (Balb/c breast cancer) cells in the metastatic model. Results: The presence of Foxp3+ regulatory T cell (Treg) correlates to the tumor proliferation (Ki-67 and Her2 expression) and higher Foxp3+ Treg prevalence and tumor Ki-67 expression inferred poorer chemotherapy response. TRAcP staining within the CD68+ macrophages were observed with better chemotherapy response. In animal model, M2 polarized macrophages have higher TRAcP expression, promote breast cancer metastasis, and attract Treg infiltrating in tumor, consistent with the clinical observation. Conclusion: TRAcP-expressing TAMs, representing M2-polarized macrophage, demonstrat local tumor-promoting and immune-suppressive effects, and chemotherapy resistance. TRAcP expression within TAM can be a potential prognostic marker for breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-07.

Abstract 2436: Endothelial integrin α3β1 stabilizes TF antigen/Galectin-3 mediated metastatic cell adhesion to the endothelium

Abstract Metastatic cell arrest in distant organ microvessels constitutes an essential rate-limiting step in hematogenous cancer metastasis. Previously, in a series of works we demonstrated the importance of interactions between cancer-associated Thomsen-Friedenreich antigen (TF-Ag) and endothelial galectin-3 (Gal-3) in facilitating the initial carbohydrate-mediated tumor cell adhesion to distant organ microvasculature. However, it was unclear which molecules stabilize TF-Ag/Gal-3 mediated adhesion and how these interactions effect subsequent events in a metastatic cascade. Here, the results of a pull down assay using TF-Ag coated gold nanoparticles followed by Western analysis identified endothelial integrin α3β1 being physically associated with TF-Ag/Gal-3 complexes. Blocking α3β1 did not affect the initial tumor cell adhesion to the endothelium, but reduced >10-fold shear force necessary to displace cancer cells stably attached to endothelial monolayers, indicating that α3β1 stabilizes tumor-endothelial cell adhesion initiated by TF-Ag/Gal-3 interactions. Subsequent Quadrupole Time-of-Flight mass spectrometry analysis revealed additional 7 proteins forming a macromolecular complexes with TF-Ag, Gal-3 and α3β1 such as filamin B, talin, IQ motif containing GTPase activating protein 1, vinculin, α actinin 4, zyxin, and T-plastin, all of which map onto focal adhesion pathway. Our data demonstrate an induction of complex signaling pathways downstream of this macromolecular complex facilitating endothelial cell retraction and metastatic cell transendothelial migration. Thus, endothelial α3β1 mobilization downstream of TF-Ag/Gal-3 interactions stabilizes tumor-endothelial cell adhesion, initiates focal adhesion formation, and induces signaling pathways in endothelial cells promoting cancer metastasis. This work was supported in part by Award Number 1I01BX000609 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development to VVG and AHA National SDG 0830287N to OVG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2436. doi:10.1158/1538-7445.AM2011-2436

DNA Hypermethylation Contributes to Incomplete Synthesis of Carbohydrate Determinants in Gastrointestinal Cancer

It has long been known that malignant transformation is associated with abnormal expression of carbohydrate determinants. The aim of this study was to clarify the cause of cancer-associated abnormal glycosylation in gastrointestinal (GI) cancers. We compared the expression levels of "glyco-genes," including glycosyltransferases and glycosidases, in normal GI mucosa and in gastric and colorectal cancer cells. To examine the possibility that DNA hypermethylation contributed to the down-regulation of these genes, we treated GI cancer cells with 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferase. The silencing of some of these glyco-genes, but not up-regulation of certain molecules, was observed. The Sd(a) carbohydrate was abundantly expressed in the normal GI mucosa, but its expression was significantly decreased in cancer tissues. When human colon and gastric cancer cells were treated with 5-aza-dC, cell surface expression of Sd(a) and the transcription of B4GALNT2, which catalyzes the synthesis of the Sd(a), were induced. The promoter region of the human B4GALNT2 gene was heavily hypermethylated in many of the GI cancer cell lines examined as well as in gastric cancer tissues (39 out of 78 cases). In addition, aberrant methylation of the B4GALNT2 gene was strongly correlated with Epstein-Barr virus-associated gastric carcinomas and occurred coincidentally with hypermethylation of the ST3GAL6 gene. Epigenetic changes in a group of glycosyltransferases including B4GALNT2 and ST3GAL6 represent a malignant phenotype of gastric cancer caused by silencing of the activity of these enzymes, which action may eventually induce aberrant glycosylation and expression of cancer-associated carbohydrate antigens.

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca2+ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell-endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell-ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell-cell contact region, and thus inhibits cell-cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca2+ -CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell-cell contact regions might be essential for this process under hypoxic stress.

P-selectin activates integrin-mediated colon carcinoma cell adhesion to fibronectin

During hematogenous cancer metastasis, tumor cells separate from a primary mass, enter the bloodstream, disperse throughout the body, migrate across vessel walls, and generate distant colonies. The later steps of metastasis superficially resemble leukocyte extravasation, a process initiated by selectin-mediated cell tethering to the blood vessel wall followed by integrin-mediated arrest and transendothelial migration. Some cancer cells express P-selectin ligands and attach to immobilized P-selectin, suggesting that these cells can arrest in blood vessels using sequential selectin- and integrin-mediated adhesion, as do leukocytes. We hypothesize that selectin binding may regulate subsequent integrin-mediated steps in metastasis. Using a model system of cultured Colo 320 human colon adenocarcinoma cells incubated with soluble P-selectin-IgG chimeric protein, we have found that P-selectin can stimulate activation of the α 5β 1 integrin resulting in a specific increase of adhesion and spreading of these cells on fibronectin substrates. P-selectin binding also induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3-K). PI3-K inhibitors blocked P-selectin-mediated integrin activation, cell attachment, and cell spreading. Inhibition of p38 MAPK activation blocked cell spreading, but not cell attachment. P-selectin binding also resulted in formation of a signaling complex containing PI3-K and p38 MAPK. These results suggest that P-selectin binding to tumor cells can activate α 5β 1 integrin via PI3-K and p38 MAPK signaling pathways leading to increased cell adhesion. We propose that P-selectin ligands are important tumor cell signaling molecules that modulate integrin-mediated cell adhesion in the metastatic process.

Morphological analysis of tumor cell/endothelial cell interactions under shear flow

In the process of hematogenous cancer metastasis, tumor cells (TCs) must shed into the blood stream, survive in the blood circulation, migrate through the vascular endothelium (extravasation) and proliferate in the target organs. However, the precise mechanisms by which TCs penetrate the endothelial cell (EC) junctions remain one of the least understood aspects of TC extravasation. This question has generally been addressed under static conditions, despite the important role of flow induced mechanical stress on the circulating cell–endothelium interactions. Moreover, flow studies were generally focused on transient or firm adhesion steps of TC–EC interactions and did not consider TCs spreading or extravasation. In this paper, we used a parallel-plate flow chamber to investigate TC–EC interactions under flow conditions. An EC monolayer was cultured on the lower plate of the flow chamber to model the endothelial barrier. Circulating TCs were introduced into the flow channel under a well-defined flow field and TC cell shape changes on the EC monolayer were followed in vitro with live phase contrast and fluorescence microscopy. Two spreading patterns were observed: radial spreading which corresponds to TC extravasation, and axial spreading where TCs formed a mosaic TC–EC monolayer. By investigating the changes in area and minor/major aspect ratio, we have established a simple quantitative basis for comparing spreading modes under various shear stresses. Contrary to radial spreading, the extent of axial spreading was increased by shear stress.

Suppression of cancer phenotypes through a multifunctional actin‐binding protein, calponin, that attacks cancer cells and simultaneously protects the host from invasion

Quantitative and/or qualitative alteration of actin cytoskeletal molecules, involved in the regulation of cellular dynamic functions, should be intimately related with cancer phenotypes. Based on several lines of experimental evidence from our group, and others, this report proposes a strategy to simultaneously attack cancer cells and protect the host from cancer invasion, with one molecule. Calponin h1, an actin-stabilizing protein that is also intimately related to signal transduction, is very often suppressed in vascular smooth muscle cells of malignant human tumors and in mesothelial cells by coexisting cancer cells. We generated mice deficient for calponin h1, exhibiting fragility in blood vessels and peritoneal membranes. Hematogenous cancer metastasis occurred more easily in the calponin h1-deficient mice than in wild-type mice, and the peritoneal dissemination was extremely enhanced. The fragility was rescued by the exogenous introduction of the calponin h1 gene into mesothelial cells of the peritoneum. Furthermore, calponin h1 gene transfer into several transformed cell lines resulted in a suppression of malignancy. The peritoneal dissemination of intraperitoneally-injected B16-F10 cells was suppressed by the calponin h1 gene, given to target both cancer cells and the mesothelial cells of the host. The multifunctional nature of the molecule, as a machinery player of cytoskeleton and mediator of signal transduction, probably resulted in a favorable recipient-discriminating effect on cancerous and normal cells. Thus, we believe that if we use adequate multifunctional molecules for therapy, it is possible to simultaneously suppress cancer phenotypes and protect normal cells from the attack of cancer cells.

Invasive human pancreatic carcinoma cells adhere to endothelial tri‐cellular corners and increase endothelial permeability

Although adhesive interactions between metastasizing cancer cells and vascular endothelial cells are critical in hematogenous metastasis, the early molecular events of the cancer-endothelial interaction remain largely obscure. Here we investigated the functional impact of cancer cells on endothelial permeability. We examined the binding of human pancreatic carcinoma cells MIA PaCa-2, PANC-1 and PSN-1 to a human umbilical vein endothelial cell (HUVEC) monolayer and the subsequent changes in the transendothelial electronic resistance (TEER) of the HUVEC. We found that MIA PaCa-2 and PANC-1 cells preferentially bound to the tri-cellular corners of HUVEC and induced a rapid and irreversible reduction of TEER. The reduction of HUVEC TEER was associated with the focal disengagement of endothelial junctional adhesion molecules VE-cadherin and CD31. Blocking antibodies to integrin beta1, CD44, or CD9 affected neither the MIA PaCa-2 binding to HUVEC nor the reduction of TEER. Specific inhibitors for metalloproteinases, tyrosine-kinases and lipoxigenases, and a neutralizing anti-vascular endothelial growth factor antibody failed to affect the MIA PaCa-2-induced reduction of HUVEC TEER, whereas treatment of the cells with paraformaldehyde or cytochalasin B abrogated the TEER reduction. These findings indicate that the MIA PaCa-2 cells bind selectively to endothelial tri-cellular corners, triggering a reduction of HUVEC TEER, which requires the active metabolism and intact actin cytoskeleton of the carcinoma cells, and is apparently unrelated to previously described cell adhesion and soluble factor pathways. Our data indicate a novel cell-contact-dependent mechanism for the cancer cell-mediated breakdown of endothelial barrier functions, which may be important in hematogenous cancer metastasis.

Sialyl Lewis antigens: association with MUC5AC protein and correlation with post-operative recurrence of non-small cell lung cancer

Sialyl Lewis antigens (sLe(x/a)) are cancer-associated carbohydrate determinants, serve as ligands of the selectin family and are associated with hematogenous metastasis of cancer. So far, the clinicopathologic values of sialyl Lewis x (sLe(x)) and sialyl Lewis a (sLe(a)) in lung cancer have remained controversial. Using immunohistochemistry, the expressions of sLe(x) and sLe(a) antigens, and an airway mucin (MUC5AC) protein, which was supposed to be the major carrying protein of sialyl Lewis moieties, were studied in surgically resected tumor tissues of 61 patients with stages I or II NSCLC. Thirty-two (52.5%) of the 61 studied subjects were found to be positive for expression of sLe(a), 40 (65.6%) were positive for expression of sLe(x), and 16 (26.2%) were positive for MUC5AC protein. Both the expression of sLe(x) and MUC5AC were associated with adenocarcinoma subtype. Patients bearing tumors with MUC5AC and/or sLe(x) expression had a higher probability of post-operative distant metastasis. Survival analysis demonstrated that patients bearing tumors with expression of sLe(x) antigen or MUC5AC had shorter overall survival. The multivariate logistic regression showed that age >65 years old (OR = 0.207, 95% CI = 0.075-0.569, P = 0.002), nodal status (OR = 6.575, 95% CI = 2.459-17.583, P < 0.001), and MUC5AC (OR = 5.545, 95% CI = 1.998-15.386, P = 0.001) were independent factors affecting survival. We concluded that the expression of sLe(x) was related to MUC5AC protein, while patients with tumors co-expressing both MUC5AC and sLe(x) antigen had the worst survival.

Loss of disialyl Lewis(a), the ligand for lymphocyte inhibitory receptor sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) associated with increased sialyl Lewis(a) expression on human colon cancers.

Expression of sialyl Lewis(a) is known to be increased in cancers of the digestive organs. The determinant serves as a ligand for E-selectin and mediates hematogenous metastasis of cancers. In contrast, disialyl Lewis(a), which has an extra sialic acid attached at the C6-position of penultimate GlcNAc in sialyl Lewis(a), is expressed preferentially on nonmalignant colonic epithelial cells, and its expression decreases significantly on malignant transformation. Introduction of the gene for an alpha2-->6 sialyl-transferase responsible for disialyl Lewis(a) synthesis to colon cancer cells resulted in a marked increase in disialyl Lewis(a) expression and corresponding decrease in sialyl Lewis(a) expression. This was accompanied by the complete loss of E-selectin binding activity of the cells. In contrast, the transfected cells acquired significant binding activity to sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7)/p75/adhesion inhibitory receptor molecule-1, an inhibitory receptor expressed on lymphoid cells. These results indicate that the transition of carbohydrate determinants from disialyl Lewis(a)-dominant status to sialyl Lewis(a)-dominant status on malignant transformation has a dual functional consequence: the loss of normal cell-cell recognition between mucosal epithelial cells and lymphoid cells on one hand and the gain of E-selectin binding activity on the other. The transcription of a gene encoding the alpha2-->6 sialyltransferase was markedly down-regulated in cancer cells compared with nonmalignant epithelial cells, which is in line with the decreased expression of disialyl Lewis(a) and increased expression of sialyl Lewis(a) in cancers. Treatment of cancer cells with butyrate or 5-azacytidine induced strongly disialyl Lewis(a) expression, suggesting that histone deacetylation and/or DNA methylation may be involved in the silencing of the gene in cancers.

Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates

Cancer cells undergo distinct metabolic changes to cope with their hypoxic environment. These changes are achieved at least partly by the action of transcriptional factors called hypoxia-inducible factors (HIFs). We investigated gene expression in cultured human colon cancer cells induced by hypoxic conditions with special reference to cell-adhesion molecules and carbohydrate determinants having cell-adhesive activity by using DNA-microarray and RT-PCR techniques. Hypoxic culture of colon cancer cells induced a marked increase in expression of selectin ligands, the sialyl Lewis x and sialyl Lewis a determinants at the cell surface, which led to a definite increase in cancer cell adhesion to endothelial E-selectin. The transcription of genes for fucosyltransferase VII (FUT7), sialyltransferase ST3Gal-I (ST3O), and UDP-galactose transporter-1 (UGT1), which are all known to be involved in the synthesis of the carbohydrate ligands for E-selectin, was significantly induced in cancer cells by hypoxic culture. In addition, a remarkable induction was detected in the genes for syndecan-4 (SDC4) and alpha5-integrin (ITGA5), the cell-adhesion molecules involved in the enhanced adhesion of cancer cells to fibronectin. The transcriptional induction by hypoxia was reproduced in the luciferase-reporter assays for these genes, which were significantly suppressed by the co-transfection of a dominant-negative form of HIF. These results indicate that the metabolic shifts of cancer cells partly mediated by HIFs significantly enhance their adhesion to vascular endothelial cells, through both selectin- and integrin-mediated pathways, and suggest that this enhancement further facilitates hematogenous metastasis of cancers and tumor angiogenesis.

Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis.

Malignant transformation is associated with abnormal glycosylation, resulting in the synthesis and expression of altered carbohydrate determinants including sialyl Lewisa and sialyl Lewisx. The sialyl Lewisa and sialyl Lewisx determinants appear in the sera of patients with cancer, and are extensively utilized for serum diagnosis of cancers in Japan. Sialyl Lewisa and sialyl Lewisx are involved in selectin-mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. Recent progress in this area includes the following: 1. Substantial increases in solid clinical statistics that further confirm the contribution of these determinants in the progression of a wide variety of cancers; 2. Elucidation of the ligand specificity of the three family members of selectins and evaluation of the roles of these molecules in cancer cell adhesion; and 3. Advances in the study of the mechanism that leads to the enhanced expression of the sialyl Lewis(a/x) determinants in malignant cells. These recent results have confirmed that these determinants are not merely markers for cancers, but are functionally implicated in the malignant behavior of cancer cells. The results also suggested that the increase of these determinants in malignant cells is an inevitable consequence of the malignant transformation of cells. Considerable new knowledge has also been accumulated regarding the therapeutic implications for suppression of hematogenous metastasis targeting this cell adhesion system.