Tumor microenvironment remodeling modulates macrophage phenotype in breast cancer lymphangiogenesis.

Hyaluronan (HA) is dynamically remodeled in tumor microenvironment (TME) and is reported to be closely related to tumor lymphatic metastasis by inducing lymphangiogenesis. Macrophages are known to be involved in neo-lymphatic vessels formation. However, few studies have investigated the role of HA-mediated TME remodeling on macrophages-dependent lymphangiogenesis. We previously showed that HA could drive macrophages to acquire the M2 phenotype. In this study, we attempt to study the crosstalk between HA in TME and macrophages dependent lymphangiogenesis. First, we found that the abundant assembly of HA in breast cancer tissue was accompanied by increased infiltration of macrophages featured by expressing lymphatic endothelial markers. Then, to further identify the remodeling of HA in regulating macrophage phenotype, we used HA fragments which are usually enriched in TME for this purpose. Our results showed that the reconstructed HA could induce bone marrow-derived macrophages (BMDMs) to express markers of lymphatic endothelium and form tube-like structures, suggesting a novel function of HA from TME on macrophages-dependent lymphangiogenesis. Finally, we found that inhibition of the HA-TLR4 pathway could reduce the ability of BMDMs to exhibit lymphatic endothelial phenotype. Our results provide new insight into tumor microenvironment remodeling and macrophages in breast cancer lymphangiogenesis.

Hyaluronan synthase 2 (HAS2) regulates cell phenotype and invadopodia formation in luminal-like breast cancer cells.

Although luminal breast cancer cells are typically highly cohesive epithelial cells and have low invasive ability, many eventually develop metastasis. Until now, the underlying mechanisms remain obscure. In this work, we showed that the level of hyaluronic acid synthase 2 (HAS2) was positively correlated with the malignant phenotype of breast cancer cells. Notably, the increased expression of HAS2 promoted the invasive and migratory abilities of luminal breast cancer cells in vitro, followed by a reduced expression of E-cadherin, ß-catenin, and ZO-1, and an elevated expression of N-cadherin and vimentin. Furthermore, overexpression of HAS2 promoted while knockdown of HAS2 impeded invadopodia formation, which subsequently increased or decreased the activation of cortactin, Tks5, and metalloproteinases (MMPs). Activation of these invadopodia-related proteins was prevented by inhibition of HAS2 or disruption of HA, which in turn attenuated the increased motility and invasiveness. Further, in vivo study showed that, HAS2 increased tumor growth and the rate of lung metastasis via driving transition to an invasive cell phenotype in SCID mice that were orthotopically transplanted with luminal breast cancer cells. Collectively, our results showed that HAS2 promoted cell invasion by inducing transition to an invasive phenotype and by enhancing invadopodia formation in luminal breast cancer cells, which may provide new mechanistic insights into its role in tumor metastasis.

CD44v6 Targeted by miR-193b-5p in the Coding Region Modulates the Migration and Invasion of Breast Cancer Cells.

Previous studies have shown that CD44 containing variant exon v6 (CD44v6) is highly expressed in many cancers and is related to tumor metastasis. However, the detailed mechanism of the regulatory pattern of CD44v6 in breast cancer remains unclear. Here, we found that CD44v6 was significantly upregulated in invasive breast cancer cell lines compared with low-invasive breast cancer cell lines. Cell migration and invasion could be suppressed by CD44v6 downregulation. MiRWalk and RNAhybrid software revealed miR-193b-5p as a miRNA targeting CD44v6 by binding to the exon v6 region. We found that the overexpression of miR-193b-5p inhibited the migration and invasion of Hs-578t and BT-549 cells, which could be rescued by restoring the expression of CD44v6. Next, we determined the potential of miR-193b-5p as an in vitro biomarker for breast cancer. Serum samples were obtained from 58 breast cancer patients, 36 patients with benign disease and 58 age-matched cancer-free controls. The results showed that the expression of miR-193b-5p in the serum was significantly lower in breast cancer patients than in controls and could distinguish cancer from cancer-free samples. The area under the receiver operating characteristic curve (ROC) for miR-193b-5p was 0.762(95% confidence interval: 0.674-0.851), which was higher than that of carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3). Combining miR-193b-5p with CEA or CA15-3 could improve the diagnostic efficiency compared with the CEA and CA15-3 combination. Taken together, our results suggest that miR-193b-5p could function as a tumor-suppressive miRNA by targeting CD44v6 in breast cancer and that serum miR-193b-5p may serve as a biomarker for breast cancer diagnosis.

Inducible formation of leader cells driven by CD44 switching gives rise to collective invasion and metastases in luminal breast carcinomas.

Collective invasion into adjacent tissue is a hallmark of luminal breast cancer, and ~20% of these cases eventually undergo metastasis. How less aggressive luminal-like breast cancer transitions to invasive cancer remains unclear. Our study revealed that CD44hi cancer cells are the leading subpopulation in collectively invading luminal cancer cells and efficiently promote the collective invasion of CD44lo/follower cells. The CD44hi/leader subpopulation showed a specific gene signature of various hybrid epithelial/mesenchymal genes and key functional coregulators of collective invasion, which was distinct from that of CD44lo/follower cells. However, the CD44hi/leader cells, which showed a partial epithelial-mesenchymal transition (EMT) phenotype, readily switched to the CD44lo phenotype along with collective migration and vice versa; this phenomenon was spontaneous and sensitive to the tumor microenvironment. The CD44lo-to-CD44hi conversion was accompanied by a shift in CD44s to CD44v but not a conversion of non-cancer stem cells to cancer stem cells (CSCs). Therefore, the CD44hi leader cells, as currently identified, are not a stable subpopulation in breast tumors. This plasticity and ability to generate CD44hi carcinoma cells with enhanced migratory and invasive behavior might be responsible for the transition from in situ to invasive behavior of luminal-type breast cancer.

Combination of plasma HA and circulating M2-like monocytes may serve as a diagnostic marker for breast cancer.

Background: Breast cancer (BC)-derived hyaluronan (HA) can induce the formation of M2-like tumor-associated macrophages (TAMs) in tumor context. However, little is known about the correlation between circulating M2-like monocytes and plasma HA in BC patients. This study focused on evaluating the relationship between circulating M2-like monocytes and plasma HA, and further appraised the diagnostic value of them in BC. Methods: The expression of M2-like TAMs and HA was determined in pathological tissues by immunohistochemistry. Flow cytometry was used to detect the levels of circulating CD14+CD204+ M2-like monocytes in 81 BC patients, 45 patients with breast benign diseases, and 46 healthy subjects. The levels of HA, CEA, and CA15-3 were measured in plasma samples using chemiluminescence method. Results: M2-like TAMs and HA expressions were elevated in BC tissues compared with benign tissues. In correspondence, the frequency of circulating CD14+CD204+ M2-like monocytes and the plasma HA levels were significantly higher in patients with BC than those in control groups. Importantly, there was a positive correlation between circulating M2-like monocytes and the plasma HA (Spearman r = 0.404, p < 0.001). Area under receiver operating characteristic curve (ROC) for the combination of circulating M2-like monocytes and HA was 0.899 (95% CI: 0.853-0.946), which was higher than the panel of CEA and CA15-3. Conclusions: The frequency of circulating CD14+CD204+ M2-like monocytes was positively correlated to plasma HA levels. The combination of circulating CD14+CD204+ M2-like monocytes and plasma HA could provide considerable diagnostic value in BC.

Inhibition of cell invasion and migration by CEACAM1-4S in breast cancer.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cell-cell adhesion molecule, has been revealed to perform an important role in tumor progression. Although there are a number of studies on CEACAM1 in patients with breast cancer, there is limited information on the roles of CEACAM1 in breast cancer metastasis. The present study aimed to identify whether CEACAM1 is involved in breast cancer development and to investigate the underlying mechanisms. First, the expression of CEACAM1 was observed in patients with breast cancer, and the association between CEACAM1 expression levels and migration and invasion of breast cancer cells was analyzed. As there are 12 isoforms of CEACAM1, of which CEACAM1-4S dominates in the human breast epithelium, subsequent study focused on CEACAM1-4S as a representative of all the isoforms. Results of the present study demonstrated that CEACAM1-4S suppresses breast cancer cell invasion and migration in a manner that is dependent on the balance between matrix metalloproteinase 2/tissue inhibitor of metalloproteinase 2 and E-/N-cadherin expression. In addition, CEACAM1-4S was likely to cause reversal of epithelial-mesenchymal transition of breast cancer cells through repressing Smad2 and signal transducer and phosphorylation of activator of transcription 3. In conclusion, the present study demonstrated that CEACAM1-4S performs an inhibitory role in breast cancer metastasis, and restoring CEACAM1-4S expression may provide a novel strategy for therapy of patients with metastatic breast cancer.

Up-regulation of carcinoembryonic antigen-related cell adhesion molecule 1 in gastrointestinal cancer and its clinical relevance.

Serum carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is dysregulated in various malignant tumors and has been associated with tumor progression. However, the expression and regulatory mechanisms of serum CEACAM1 in gastrointestinal cancer are still unclear. The expression ratio of the CEACAM1-L and CEACAM1-S isoforms has seldom been investigated in gastrointestinal cancer. In this study, we intended to explore the expression and diagnostic value of CEACAM1 in gastrointestinal cancer. Serum CEACAM1 levels were measured by enzyme-linked immunosorbent assay. The protein expression and distribution of CEACAM1 in tumors were examined by immunohistochemical staining. The expression patterns and ratio of CEACAM1-L/S were analyzed by reverse transcription-polymerase chain reaction. The results showed that serum CEACAM1 levels were significantly higher in cancer patients than in healthy controls. CEACAM1 was found in secreted forms within the neoplastic glands, and its expression was more intense at the tumor invasion front. The CEACAM1-L/S (L:S) ratios were up-regulated during tumorigenesis. Our data suggest that the serum level of CEACAM1 may be used to discriminate gastrointestinal cancer patients from health controls.

Increased circulating M2-like monocytes in patients with breast cancer.

M2-like tumor-associated macrophages promote breast tumor growth and survival and may migrate into the peripheral blood. However, the frequency of circulating M2-like monocytes in the peripheral blood of breast cancer patients has not been clarified. The objective of this study was to determine the percentages of circulating M2-like monocytes in patients with breast cancer. Immunofluorescence staining for CD68 and CD163 was performed to detect M2-like macrophages in pathological tissues. Flow cytometry was used to assess the frequencies of circulating CD14+CD163+/CD14+CD204+/CD14+CD163+CD204+ M2-like monocytes in 99 breast cancer patients, 56 patients with benign breast disease, and 60 healthy controls. Receiver operating characteristic curve analysis was used to compare the diagnostic values of circulating M2-like monocytes, carcinoembryonic antigen, and cancer antigen 15-3. The associations among circulating M2-like monocytes and clinical breast cancer parameters were analyzed. The number of CD68+CD163+ M2-like macrophages was significantly higher in breast cancer tissues than in benign tissues. In the peripheral blood, CD14+CD163+/CD14+CD204+/CD14+CD163+CD204+ M2-like monocytes were elevated in breast cancer patients compared with normal controls and patients with benign breast disease. The area under the receiver operating curve for circulating CD14+CD163+CD204+ M2-like monocytes was 0.888 (95% confidence interval: 0.839-0.936), a value higher than those for carcinoembryonic antigen and cancer antigen 15-3. High frequencies of circulating CD14+CD204+ and CD14+CD163+CD204+ M2-like monocytes were associated with tumor-node-metastasis stage, lymph node metastasis, histological differentiation, and estrogen receptor expression. Circulating M2-like monocytes may serve as a diagnostic biomarker in breast cancer and have a potential role in reflecting breast cancer progression.

Low-molecular-weight hyaluronan (LMW-HA) accelerates lymph node metastasis of melanoma cells by inducing disruption of lymphatic intercellular adhesion.

Endothelial integrity defects initiate lymphatic metastasis of tumor cells. Low-molecular-weight hyaluronan (LMW-HA) derived from plasma and interstitial fluid was reported to be associated with tumor lymphatic metastasis. In addition, LMW-HA was proved to disrupt lymphatic vessel endothelium integrity, thus promoting lymphatic metastasis of tumor cells. Until now, there are few reports on how LMW-HA modulates lymphatic endothelial cells adhesion junctions and affects cancer cells metastasizing into lymph vessels. The aim of our study is to unravel the novel mechanism of LMW-HA in mediating tumor lymphatic metastasis. Here, we employed a melanoma metastasis model to investigate whether LMW-HA facilitates tumor cells transferring from foci to remote lymph nodes by disrupting the lymphatic endothelial integrity. Our data indicate that LMW-HA significantly induces metastasis of melanoma cells to lymph nodes and accelerates interstitial-lymphatic flow in vivo. Further experiments show that increased migration of melanoma cells across human dermal lymphatic endothelial cell (HDLEC) monolayers is accompanied by impaired lymphatic endothelial barrier function and increased permeability. The mechanism study reveals that VE-cadherin-ß-catenin pathway and relevant signals are involved in modulating the interactions between endothelial cells and that a significant inhibition of lymphatic endothelium disruption is observed when antibodies to the LMW-HA receptor (LYVE-1) are present. Thus, our findings demonstrate a disruptive effect of LMW-HA on lymphatic endothelium continuity which leads to a promotion on melanoma lymphatic metastasis and also suggest a cellular signaling mechanism associated with VE-cadherin-mediated lymphatic intercellular junctions.

A novel role of low molecular weight hyaluronan in breast cancer metastasis.

Low molecular weight hyaluronan (LMW-HA), a degradation fragment of the extracellular matrix component hyaluronan (HA), has been proven to play a crucial role in cancer progression. However, no systematic clinical study of breast cancer has been performed to correlate LMW-HA levels with metastasis. In the present study, we analyzed 176 serum specimens and found for the first time that the serum LMW-HA (but not total HA) level significantly correlated with lymph node metastasis, suggesting that serum LMW-HA represents a better prognostic indicator of breast cancer progression than HA. Similarly, we found that breast cancer cell lines displaying higher invasive potential had a higher LMW-HA concentration than less-invasive cell lines. This higher LMW-HA level was accompanied by the overexpression of hyaluronan synthase (HAS2) and hyaluronidase (both HYAL1 and HYAL2). Of great importance, decreasing LMW-HA production significantly inhibited breast cancer cell migration and invasion. Overall, our results suggest that during cancer progression, cancer cells may actively remodel their microenvironment via an autocrine/paracrine-like process, resulting in elevated LMW-HA levels, which in turn may facilitate cancer progression by promoting the migration and invasion of cancer cells. Therefore, cancer-associated LMW-HA may be a more promising molecular biomarker than total HA for detecting metastasis and may have further applications in breast cancer treatment.

Carpal tunnel syndrome assessment with ultrasonography: value of inlet-to-outlet median nerve area ratio in patients versus healthy volunteers.

OBJECTIVE: To evaluate the diagnostic value of the Inlet-to-outlet median nerve area ratio (IOR) in patients with clinically and electrophysiologically confirmed carpal tunnel syndrome (CTS). METHODS: Forty-six wrists in 46 consecutive patients with clinical and electrodiagnostic evidence of CTS and forty-four wrists in 44 healthy volunteers were examined with ultrasonography. The cross-sectional area (CSA) of the median nerve was measured at the carpal tunnel inlet (the level of scaphoid-pisiform) and outlet (the level of the hook of the hamate), and the IOR was calculated for each wrist. Ultrasonography and electrodiagnostic tests were performed under blinded conditions. Electrodiagnostic testing combined with clinical symptoms were considered to be the gold standard test. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value between the inlet CSA and IOR. RESULTS: The study population included 16 men and 30 women (mean age, 45.3 years; range, 18-83 years). The control population included 18 men and 26 women (mean age, 50.4 years; range, 18-79 years). The mean inlet CSA was 8.7 mm2 in healthy controls and 14.6mm2 in CTS group (P<0.001). The mean IOR in healthy volunteers (1.0) was smaller than that in patients (1.6, P<0.001). Receiver operating characteristic analysis revealed a diagnostic advantage to using the IOR rather than the inlet CSA (P<0.01). An IOR cutoff value of ≥ 1.3 would yield 93% specificity and 91% sensitivity in the diagnosis of CTS. CONCLUSION: The IOR of median nerve area promises to be an effective means in the diagnosis of CTS. A large-scale, randomized controlled trial is required to determine how and when this parameter will be used.

Comparative study of microRNA profiling in keloid fibroblast and annotation of differential expressed microRNAs.

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. The molecular mechanism underlying keloid pathogenesis is still largely unknown. In this study, we compared microRNA (miRNA) expression profiles between keloid-derived fibroblasts and normal fibroblasts (including fetal and adult dermal fibroblasts) by miRNA microarray analysis. We found that the miRNA profiles in keloid-derived fibroblasts are different with those in normal fibroblasts. Nine miRNAs were differentially expressed, six of which were significantly up-regulated in keloid fibroblasts (KFs), including miR-152, miR-23b-3p, miR-31-5p, miR-320c, miR-30a-5p, and hsv1-miR-H7, and three of which were significantly down-regulated, including miR-4328, miR-145-5p, and miR-143-3p. Functional annotations of differentially expressed miRNA targets revealed that they were enriched in several signaling pathways important for scar wound healing. In conclusion, we demonstrate that the miRNA expression profile is altered in KFs compared with in fetal and adult dermal fibroblasts, and the expression profile may provide a useful clue for exploring the pathogenesis of keloids. miRNAs might partially contribute to the etiology of keloids by affecting several signaling pathways relevant to scar wound healing.

The high and low molecular weight forms of hyaluronan have distinct effects on CD44 clustering.

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment.

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

Preparation and characterization of hyaluronan oligosaccharides for angiogenesis study.

Low molecular weight hyaluronan (HA) is proved to be active in stimulating endothelial cell (EC) proliferation. The study is to obtain biological active HA fragments of defined length from high molecular weight HA for angiogenesis research. A novel method, by a high-voltage polyacrylamide-gel electrophoresis (PAGE) that uses specific fluorophore labeling, was developed to identify the degraded products digested from native high molecular weight HA. The polymer HA was digested by hyaluornidase partially, and the enzymatic fragments of HA or HA oligosaccharides (o-HAs) were analyzed for molecular sizes by a combination of PAGE, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The purified HA oligomers were pooled and the biological effects of the mixtures on EC proliferation were studied, by separating the digests of HA fragments by size exclusion chromatography on a Bio-Gel P-10 column. The prepared o-HAs were collected and analyzed by a series of methods, PAGE, HPLC, and MS, which indicated a mixture of oligosaccharides covering a range of different sizes from 4 to 20-mer. The minimal molecular size determined by the special PAGE was 2 disaccharide, and the method can determine o-HA mass as high as 10 disaccharide (seeing the least clear band as much as possible on gel). Fractions containing HA fragments with sizes from 4 to 20-mer were pooled by distinct oligosaccharide species and added to the culture of pig endothelial cell (PIEC) for biological activities study. A significant increase in EC proliferation was found in comparison with nondigested high molecular weight HA. Our results suggested that o-HA can be obtained by a method of fluorophore-labeled olyacrylamide gel electrophoresis for identifying as small as 4-mer oligomer HA from high molecular weight HA-digested products. This method was proved to be sensitive, simple, and rapid in characterizing saccharides, which may be used by replacing the complicated ways of HPLC and MS. The prepared o-HA was found to be active in stimulating EC proliferation.

Brain hyaluronan binding protein inhibits tumor growth.

BACKGROUND: Great efforts have been made to search for the angiogenic inhibitors in avascular tissues. Several proteins isolated from cartilage have been proved to have anti-angiogenic or anti-tumour effects. Because cartilage contains a great amount of hyaluronic acid (HA) oligosaccharides and abundant HA binding proteins (HABP), therefore, we speculated that HABP might be one of the factors regulating vascularization in cartilage or anti-angiogenesis in tumours. The purpose of this research was to evaluate the effects of hyaluronan binding protein on inhibiting tumour growth both in vivo and vitro. METHODS: A unique protein termed human brain hyaluronan (HA) binding protein (b-HABP) was cloned from human brain cDNA library. MDA-435 human breast cancer cell line was chosen as a transfectant. The in vitro underlying mechanisms were investigated by determining the possibilities of MDA-435/b-HABP colony formation on soft agar, the effects of the transfectant on the proliferation of endothelial cells and the expression levels of caspase 3 and FasL from MDA-435/b-HABP. The in vivo study included tumour growth on the chorioallantoic membrane (CAM) of chicken embryos and nude mice. RESULTS: Colony formation assay revealed that the colonies formed by MDA-435/b-HABP were greatly reduced compared to mock transfectants. The conditioned media from MDA-435/b-HABP inhibited the growth of endothelial cells in culture. Caspase 3 and FasL expressions were induced by MDA-435/b-HABP. The size of tumours of MDA-435/b-HABP in both CAM and nude mice was much smaller than that of MDA-435 alone. CONCLUSIONS: Human brain hyaluronan binding protein (b-HABP) may represent a new kind of naturally existing anti-tumour substance. This brain-derived glycoprotein may block tumour growth by inducing apoptosis of cancer cells or by decreasing angiogenesis in tumour tissue via inhibiting proliferation of endothelial cells.