Gene Summary

Gene:HAS2; hyaluronan synthase 2
Summary: Hyaluronan or hyaluronic acid (HA) is a high molecular weight unbranched polysaccharide synthesized by a wide variety of organisms from bacteria to mammals, and is a constituent of the extracellular matrix. It consists of alternating glucuronic acid and N-acetylglucosamine residues that are linked by beta-1-3 and beta-1-4 glycosidic bonds. HA is synthesized by membrane-bound synthase at the inner surface of the plasma membrane, and the chains are extruded through pore-like structures into the extracellular space. It serves a variety of functions, including space filling, lubrication of joints, and provision of a matrix through which cells can migrate. HA is actively produced during wound healing and tissue repair to provide a framework for ingrowth of blood vessels and fibroblasts. Changes in the serum concentration of HA are associated with inflammatory and degenerative arthropathies such as rheumatoid arthritis. In addition, the interaction of HA with the leukocyte receptor CD44 is important in tissue-specific homing by leukocytes, and overexpression of HA receptors has been correlated with tumor metastasis. HAS2 is a member of the newly identified vertebrate gene family encoding putative hyaluronan synthases, and its amino acid sequence shows significant homology to glycosaminoglycan synthetase (DG42) from Xenopus laevis, and human and murine hyaluronan synthase 1. [provided by RefSeq, Jul 2008]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:hyaluronan synthase 2
Source:NCBIAccessed: 15 March, 2017


What does this gene/protein do?
Show (13)

Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 15 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Tumor Microenvironment
  • Transfection
  • Glucuronosyltransferase
  • Signal Transduction
  • Cell Cycle
  • Cell Movement
  • Extracellular Matrix
  • Neoplasm Metastasis
  • Cell Proliferation
  • Uridine Diphosphate Glucuronic Acid
  • Transcriptome
  • DNA-Binding Proteins
  • Disease Progression
  • Promoter Regions
  • HAS2
  • Hymecromone
  • Neoplasm Invasiveness
  • Sensitivity and Specificity
  • Transferases
  • Antigens, CD44
  • Breast Cancer
  • Ovarian Cancer
  • Zinc Fingers
  • Bone Cancer
  • Infant
  • Oncogene Fusion Proteins
  • Biomarkers, Tumor
  • Chromosome 8
  • Cell Adhesion
  • Hyaluronoglucosaminidase
  • Versicans
  • Cancer Gene Expression Regulation
  • Transforming Growth Factor beta
  • Spectral Karyotyping
  • Up-Regulation
  • Sialoglycoproteins
  • Base Sequence
  • Apoptosis
  • beta Catenin
  • Hyaluronic Acid
Tag cloud generated 15 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (3)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: HAS2 (cancer-related)

Zhang HY, Liang F, Wang F, et al.
In Vitro Effects of HAS-2 Gene Silencing on the Proliferation and Apoptosis of the MCF-7 Human Breast Cancer Cell Line.
Cell Physiol Biochem. 2016; 40(3-4):807-817 [PubMed] Related Publications
BACKGROUND: Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. This study is aimed to investigate the effects of silencing the HAS-2 gene on the proliferation and apoptosis of human breast cancer cells.
METHODS: MCF-7 cells were collected and assigned into control, scrambled siRNA and HAS-2- siRNA groups. After transfection, the morphological changes in the MCF-7 cells were observed using phase contrast microscopy. qRT-PCR and Western blot assays were used to detect the mRNA and protein expression of apoptosis-related proteins. CCK-8 and flow cytometry were performed to evaluate cell proliferation, the cell cycle and apoptosis.
RESULTS: In the control and the scrambled siRNA groups, cells grew adhered to the wall and mainly showed a spindle shape with a clear nucleolus. Compared with the control and scrambled siRNA groups, increases in the number of cells in early apoptosis and metaphase cells in apoptosis were observed in the HAS-2-siRNA group. The HAS-2-siRNA group showed decreased expression of HAS-2 relative to that in the control and scrambled siRNA groups. No significant differences in cell proliferation, cell cycle distribution or apoptosis were noted between the control and scrambled siRNA groups. In the HAS-2-siRNA group, the cell proliferation ability decreased significantly, but the number of cells in the G0/G1 stage, the number of apoptotic cells and the expression of caspase-3 and caspase-9 increased significantly.
CONCLUSION: Our findings indicate that HAS-2 gene silencing may inhibit proliferation and promote apoptosis in the MCF-7 human breast cancer cell line.

Zhang H, Tsang JY, Ni YB, et al.
Hyaluronan synthase 2 is an adverse prognostic marker in androgen receptor-negative breast cancer.
J Clin Pathol. 2016; 69(12):1055-1062 [PubMed] Related Publications
AIMS: The important role of hyaluronan synthase 2 (HAS2), an isozyme responsible for hyaluronan synthesis, in cancer has been increasingly recognised. However, only a few studies with inconsistent results have been reported in breast cancers. With a large cohort, we aim to determine the clinical significance of HAS2 in breast cancers.
METHODS: We examined HAS2 expression in 1142 breast cancers using immunohistochemistry.
RESULTS: HAS2 was associated with both prognostically favourable (androgen receptor (AR), p<0.001) and unfavourable (basal and epithelial mesenchymal transition markers, p≤0.039) biomarkers. In addition, HAS2 showed differential associations with various features and outcome between AR+ and AR- subgroups. HAS2+AR- breast cancers showed significantly worse outcome than other subgroups, and HAS2+AR- subgroup was an independent adverse prognostic factor for disease-free survival (HR 1.309, p=0.046). Interestingly, HAS2 was associated with many poor prognostic features (including higher grade, lymphovascular invasion, basal-like breast cancer subtype, high Ki67 and basal marker expression) only in AR-, but not AR+ breast cancers.
CONCLUSIONS: HAS2 has been proposed to be a target for therapeutic intervention in cancer. Our findings suggested a possible antagonistic role of AR pathway on HAS2 function. It will be interesting to further investigate their precise interaction, which may have important implication in HAS2 targeting.

Li P, Xiang T, Li H, et al.
Hyaluronan synthase 2 overexpression is correlated with the tumorigenesis and metastasis of human breast cancer.
Int J Clin Exp Pathol. 2015; 8(10):12101-14 [PubMed] Free Access to Full Article Related Publications
Extracellular matrix (ECM) is closely correlated with the malignant behavior of breast cancer cells. Hyaluronan (HA) is one of the main components of ECM, and actively regulates cell adhesion, migration and proliferation by interacting with specific cell surface receptors such as CD44 and RHAMM. HA synthase 2 (HAS2) catalyzes the synthesis of HA, but its role in breast tumorigenesis remains unclear. This study assessed the roles of HAS2 in malignant behavior of human breast cancer and sought to provide mechanistic insights into the biological and pivotal roles of HAS2. We observed HAS2 was overexpressed in breast cancer cell lines and invasive duct cancer tissues, compared with the nonmalignant breast cell lines and normal breast tissues. In addition, a high level of HAS2 expression was statistically correlated with lymph node metastasis. Functional assays showed that knockdown of HAS2 expression inhibited breast tumor cell proliferation in vivo and in vitro, through the induction of apoptosis or cell cycle arrest. Further studies showed that the HA were elevated in breast cancer, and HAS2 could upregulate HA expression. In conclusion, HAS2-HA system influences the biological characteristics of human breast cancer cells, and HAS2 may be a potential prognostic marker and therapeutic target in breast cancer.

Katona É, Juhász T, Somogyi CS, et al.
PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.
Int J Oncol. 2016; 48(3):983-97 [PubMed] Free Access to Full Article Related Publications
Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

Warren M, Turpin BK, Mark M, et al.
Undifferentiated myxoid lipoblastoma with PLAG1-HAS2 fusion in an infant; morphologically mimicking primitive myxoid mesenchymal tumor of infancy (PMMTI)--diagnostic importance of cytogenetic and molecular testing and literature review.
Cancer Genet. 2016 Jan-Feb; 209(1-2):21-9 [PubMed] Related Publications
Lipoblastoma is a benign myxoid neoplasm arising in young children that typically demonstrates adipose differentiation. It is often morphologically indistinguishable from primitive myxoid mesenchymal tumor of infancy (PMMTI), which is characterized by a well-circumscribed myxoid mass with a proliferation of primitive mesenchymal cells with mild cytologic atypia. PMMTI occurs in the first year of life and is known to have locally aggressive behavior. No specific genetic rearrangements have been reported to date. In contrast, the presence of PLAG1 (Pleomorphic Adenoma Gene 1) rearrangement is diagnostic for lipoblastoma. We hereby demonstrate the combined application of multiple approaches to tackle the diagnostic challenges of a rapidly growing neck tumor in a 3-month-old female. An incisional tumor biopsy had features of an undifferentiated, myxoid mesenchymal neoplasm mimicking PMMTI. However, tumor cells showed diffuse nuclear expression by immunohistochemical (IHC) stain. Conventional cytogenetic and fluorescence in situ hybridization (FISH) analyses as well as next generation sequencing (NGS) demonstrated evidence of PLAG1 rearrangement, confirming the diagnosis of lipoblastoma. This experience warrants that undifferentiated myxoid lipoblastoma can mimic PMMTI, and the combination of cytogenetic and molecular approaches is essential to distinguish these two myxoid neoplasms. Literature on lipoblastomas with relevant molecular and cytogenetic findings is summarized. Our case is the first lipoblastoma diagnosed with a PLAG1 fusion defined by NGS technology.

Kohi S, Sato N, Cheng XB, et al.
A novel epigenetic mechanism regulating hyaluronan production in pancreatic cancer cells.
Clin Exp Metastasis. 2016; 33(3):225-30 [PubMed] Related Publications
Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma enriched with hyaluronan (HA), a major component of extracellular matrix known to play a critical role in tumor progression. The mechanisms that regulate HA synthesis in PDAC are poorly understood. To investigate whether DNA methylation and HA production from PDAC cells are associated, we studied the effect of 5-aza-2'-deoxycitidine (5-aza-dC), an inhibitor of DNA methylation, or DNA methyltransferase 1 (DNMT1) knockdown by small interfering RNA, on the HA production from PDAC cells. HA production into the conditioned medium was evaluated in PDAC cells treated with 5-aza-dC or DNMT1 knockdown. mRNA expression of HA synthase (HAS) genes was investigated by real-time RT-PCR. Treatment of PDAC cells with 5-aza-dC led to a significant increase in the HA production (up to 2.5-fold increase) in all 4 cell lines tested. This enhanced HA production by 5-aza-dC treatment was accompanied by increased mRNA expression of HAS2 and HAS3. Furthermore, increased HA production and HAS2/HAS3 mRNA expression was also observed in PDAC cells by knockdown of DNMT1. These findings provide evidence, for the first time, that epigenetic mechanism is involved in the regulation of HA synthesis in PDAC cells.

Guin S, Ru Y, Agarwal N, et al.
Loss of Glycogen Debranching Enzyme AGL Drives Bladder Tumor Growth via Induction of Hyaluronic Acid Synthesis.
Clin Cancer Res. 2016; 22(5):1274-83 [PubMed] Free Access to Full Article Related Publications
PURPOSE: We demonstrated that amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL) is a tumor growth suppressor and prognostic marker in human bladder cancer. Here we determine how AGL loss enhances tumor growth, hoping to find therapeutically tractable targets/pathways that could be used in patients with low AGL-expressing tumors.
EXPERIMENTAL DESIGN: We transcriptionally profiled bladder cell lines with different AGL expression. By focusing on transcripts overexpressed as a function of low AGL and associated with adverse clinicopathologic variables in human bladder tumors, we sought to increase the chances of discovering novel therapeutic opportunities.
RESULTS: One such transcript was hyaluronic acid synthase 2 (HAS2), an enzyme responsible for hyaluronic acid (HA) synthesis. HAS2 expression was inversely proportional to that of AGL in bladder cancer cells and immortalized and normal urothelium. HAS2-driven HA synthesis was enhanced in bladder cancer cells with low AGL, and this drove anchorage-dependent and independent growth. siRNA-mediated depletion of HAS2 or inhibition of HA synthesis by 4-methylumbelliferone (4MU) abrogated in vitro and xenograft growth of bladder cancer cells with low AGL. AGL and HAS2 mRNA expression in human tumors was inversely correlated in patient datasets. Patients with high HAS2 and low AGL tumor mRNA expression had poor survival, lending clinical support to xenograft findings that HAS2 drives growth of tumors with low AGL.
CONCLUSIONS: Our study establishes HAS2-mediated HA synthesis as a driver of growth of bladder cancer with low AGL and provides preclinical rationale for personalized targeting of HAS2/HA signaling in patients with low AGL-expressing tumors.

Hofvander J, Tayebwa J, Nilsson J, et al.
RNA sequencing of sarcomas with simple karyotypes: identification and enrichment of fusion transcripts.
Lab Invest. 2015; 95(6):603-9 [PubMed] Related Publications
Gene fusions are neoplasia-associated mutations arising from structural chromosomal rearrangements. They have a strong impact on tumor development and constitute important diagnostic markers. Malignant soft tissue tumors (sarcomas) constitute a heterogeneous group of neoplasms with >50 distinct subtypes, each of which is rare. In addition, there is considerable morphologic overlap between sarcomas and benign lesions. Several subtypes display distinct gene fusions, serving as excellent biomarkers. The development of methods for deep sequencing of the complete transcriptome (RNA-Seq) has substantially improved the possibilities for detecting gene fusions. With the aim of identifying new gene fusions of biological and clinical relevance, eight sarcomas with simple karyotypes, ie, only one or a few structural rearrangements, were subjected to massively parallel paired-end sequencing of mRNA. Three different algorithms were used to identify fusion transcripts from RNA-Seq data. Three novel (KIAA2026-NUDT11, CCBL1-ARL1, and AFF3-PHF1) and two previously known fusions (FUS-CREB3L2 and HAS2-PLAG1) were found and could be verified by other methods. These findings show that RNA-Seq is a powerful tool for detecting gene fusions in sarcomas but also suggest that it is advisable to use more than one algorithm to analyze the output data as only two of the confirmed fusions were reported by more than one of the gene fusion detection software programs. For all of the confirmed gene fusions, at least one of the genes mapped to a chromosome band implicated by the karyotype, suggesting that sarcomas with simple karyotypes constitute an excellent resource for identifying novel gene fusions.

Tamura R, Yokoyama Y, Yoshida H, et al.
4-Methylumbelliferone inhibits ovarian cancer growth by suppressing thymidine phosphorylase expression.
J Ovarian Res. 2014; 7:94 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: 4-Methylumbelliferone (4-MU), a hyaluronan (HA) synthesis inhibitor, has antitumor activity in cancer cells. However, few studies have focused on its effects on ovarian cancer. The aim of this study was to investigate the effects of 4-MU on ovarian cancer and to elucidate its mechanism of action.
METHODS: The HRA human ovarian serous adenocarcinoma cell line was used in this study. The effects of 4-MU on cell proliferation, migration, and invasion were determined by using in vitro assays as well as an in vivo rat peritoneal carcinomatosis model. The expression of HA synthase (HAS), CD44 HA receptor, vascular endothelial growth factor (VEGF), and thymidine phosphorylase (TP) mRNA in HRA cells was analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).
RESULTS: 4-MU administration inhibited the growth of peritoneal tumors and significantly prolonged survival. In vitro experiments showed that 4-MU inhibited HRA cell proliferation in a dose-dependent manner, while it did not affect HRA cell invasion and migration. 4-MU significantly decreased TP mRNA expression in HRA cells. On the other hand, since HAS2, CD44, and VEGF endogenous mRNA expression levels were very low in HRA cells, it was impossible to evaluate the effect of 4-MU treatment.
CONCLUSIONS: These results suggest that 4-MU exerts its antitumor effect on ovarian cancer through suppressing TP expression.

Heldin P, Basu K, Kozlova I, Porsch H
HAS2 and CD44 in breast tumorigenesis.
Adv Cancer Res. 2014; 123:211-29 [PubMed] Related Publications
Metastatic spread of breast cancer cells, facilitated by the epithelial-mesenchymal transition (EMT) process, is responsible for the majority of breast cancer mortality. Increased levels of hyaluronan due to deregulation of hyaluronan-synthesizing enzymes, like HAS2, and expression of CD44, the key receptor for hyaluronan, are correlated to poor outcome of patients with basal-like breast cancer. TGFβ induces HAS2 and CD44, both of which are required in the course of efficient TGFβ-induced EMT processes by mammary epithelial cells. Elucidation of the molecular mechanisms underlying tumor-stroma interactions in breast cancer including the regulation of HAS2 and CD44 expression may contribute to the development of better strategies to treat breast cancer patients.

Vigetti D, Passi A
Hyaluronan synthases posttranslational regulation in cancer.
Adv Cancer Res. 2014; 123:95-119 [PubMed] Related Publications
Hyaluronan (HA) is a critical component of cancer microenvironment that is known to increase tumor progression and aggressiveness. The synthesis of HA starts from the cytosolic precursors UDP-N-acetylglucosamine and UDP-glucuronic acid. These two sugar nucleotides have several functions in addition to glycoconjugate synthesis and glucuronidation reactions, each of which can have a critical role in cancer. HA is synthesized by a family of three HA synthase (HAS) enzymes and, in this review, we described the main posttranslational modifications that are known to regulate HA metabolism. In particular, as the main HAS in adult tissues is HAS2, we focused on the role of AMPK-mediated phosphorylation and glycosylation by O-linked N-acetylglucosamine (O-GlcNAcylation) of HAS2 which mediate HAS2 inactivation and activation, respectively. HA catabolism, furnishing glucuronic acid and N-acetylglucosamine, can represent for a cancer cell a valid source of substrates to sustain complex tumor metabolism, and we highlight a presumable metabolic fate of such sugars in tumor cells.

Chanmee T, Ontong P, Mochizuki N, et al.
Excessive hyaluronan production promotes acquisition of cancer stem cell signatures through the coordinated regulation of Twist and the transforming growth factor β (TGF-β)-Snail signaling axis.
J Biol Chem. 2014; 289(38):26038-56 [PubMed] Free Access to Full Article Related Publications
The cancer stem cell (CSC) model suggests that a small subpopulation of cancer cells possesses the ability to self-renew and give rise to malignant progeny that drive cancer progression. Recent reports have also proposed the existence of certain extra- or intracellular signals that allow cancer progenitors to dynamically revert to a stem cell state. However, the mechanisms underlying cancer cell plasticity and CSC expansion are not entirely clear. Our previous studies using a hyaluronan synthase 2 (Has2) transgenic mouse model demonstrated that hyaluronan overproduction caused rapid development of aggressive breast carcinoma at a high incidence. Thus, we hypothesize that hyaluronan overproduction may accelerate cancer progression by expanding CSC subpopulations during cancer development. Primary cancer cells were established from mammary tumors developed in the transgenic mice and subjected to the Hoechst 33342 dye exclusion assay to sort side population (SP) from non-side population (non-SP) cells. Flow cytometric analysis demonstrated the enrichment of CD44(high)/CD24(low) CSC-like cells in the SP fraction of hyaluronan-overproducing cancer cells. This subpopulation exhibited several characteristics that were similar to CSCs, including cancer-initiating and mammosphere-forming abilities. Excess hyaluronan production drove the epithelial-to-mesenchymal transition process defined as the loss of epithelial phenotypes, up-regulation of transforming growth factor β (TGF-β), and induction of the epithelial-to-mesenchymal transition-related transcriptional factors Snail and Twist. Inhibition of TGF-β-Snail signaling or silencing of Twist expression abrogated the entrance into a stem cell state. Taken together, our findings suggest that hyaluronan overproduction allows plastic cancer cell populations to revert to stem cell states via Twist and the TGF-β-Snail signaling axis.

Yoshida H, Miyachi M, Ouchi K, et al.
Identification of COL3A1 and RAB2A as novel translocation partner genes of PLAG1 in lipoblastoma.
Genes Chromosomes Cancer. 2014; 53(7):606-11 [PubMed] Related Publications
Lipoblastoma is a rapidly growing, benign neoplasm in children. Surgical excision is usually curative, with a recurrence rate of about 20%. Because the histology of lipoblastoma is heterogeneous and overlaps with other lipomatous tumors, some lipoblastoma cases have been difficult to diagnose. The detection of PLAG1 gene rearrangement is useful for the diagnosis of lipoblastoma. Three fusion partner genes are known in relation to PLAG1 in lipoblastoma HAS2 at 8q24.1, COL1A2 at 7q22, and RAD51L1 at 14q24. Herein, we describe another two novel fusion genes in lipoblastoma tumor specimens. We checked six tumors for the presence of two known fusion genes, HAS2-PLAG1 and COL1A2-PLAG1. Only HAS2-PLAG1 was found in one of the cases. Next, we attempted to identify potential PLAG1 fusion partners using 5'RACE. Sequence analysis revealed two novel fusion genes, COL3A1-PLAG1 in three cases and RAB2A-PLAG1 in one case, respectively. As a result of the translocations, the constitutively active promoter of the partner gene drives the ectopic expression of PLAG1. We also evaluated whether a high level of PLAG1 expression can be used to help differentiate lipomatous tumors. PLAG1 expression was evaluated by real-time PCR in five lipoblastoma tumor specimens. The expressions were 70-150 times higher in lipoblastomas than in human adipocytes. However, PLAG1 expression was low in one case of lipoma. These results demonstrate that PLAG1 overexpression is a potential marker of lipoblastoma. Our findings, in agreement with previous studies, show that lipoblastoma is a group of lipomatous tumors with PLAG1 rearrangement and overexpression. © 2014 Wiley Periodicals, Inc.

Gialeli Ch, Viola M, Barbouri D, et al.
Dynamic interplay between breast cancer cells and normal endothelium mediates the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells.
Biochim Biophys Acta. 2014; 1840(8):2549-59 [PubMed] Related Publications
BACKGROUND: Breast cancer-endothelium interactions provide regulatory signals facilitating tumor progression. The endothelial cells have so far been mainly viewed in the context of tumor perfusion and relatively little is known regarding the effects of such paracrine interactions on the expression of extracellular matrix (ECM), proteasome activity and properties of endothelial cells.
METHODS: To address the effects of breast cancer cell (BCC) lines MDA-MB-231 and MCF-7 on the endothelial cells, two cell culture models were utilized; one involves endothelial cell culture in the presence of BCCs-derived conditioned media (CM) and the other co-culture of both cell populations in a Transwell system. Real-time PCR was utilized to evaluate gene expression, an immunofluorescence assay for proteasome activity, and functional assays (migration, adhesion and invasion) and immunofluorescence microscopy for cell integrity and properties.
RESULTS: BCC-CM decreases the cell migration of HUVEC. Adhesion and invasion of BCCs are favored by HUVEC and HUVEC-CM. HA levels and the expression of CD44 and HA synthase-2 by HUVEC are substantially upregulated in both cell culture approaches. Adhesion molecules, ICAM-1 and VCAM-1, are also highly upregulated, whereas MT1-MMP and MMP-2 expressions are significantly downregulated in both culture systems. Notably, the expression and activity of the proteasome β5 subunit are increased, especially by the action of MDA-MB-231-CM on HUVEC.
CONCLUSIONS AND GENERAL SIGNIFICANCE: BCCs significantly alter the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells. Deep understanding of such paracrine interactions will help to design novel drugs targeting breast cancer at the ECM level. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

LaPorta E, Welsh J
Modeling vitamin D actions in triple negative/basal-like breast cancer.
J Steroid Biochem Mol Biol. 2014; 144 Pt A:65-73 [PubMed] Free Access to Full Article Related Publications
Breast cancer is a heterogeneous disease with six molecularly defined subtypes, the most aggressive of which are triple negative breast cancers that lack expression of estrogen receptor (ER) and progesterone receptor (PR) and do not exhibit amplification of the growth factor receptor HER2. Triple negative breast cancers often exhibit basal-like gene signatures and are enriched for CD44+ cancer stem cells. In this report we have characterized the molecular actions of the VDR in a model of triple negative breast cancer. Estrogen independent, invasive mammary tumor cell lines established from wild-type (WT) and VDR knockout (VDRKO) mice were used to demonstrate that VDR is necessary for 1,25-dihydroxyvitamin D3 (1,25D) mediated anti-cancer actions in vitro and to identify novel targets of this receptor. Western blotting confirmed differential VDR expression and demonstrated the lack of ER, PR and Her2 in these cell lines. Re-introduction of human VDR (hVDR) into VDRKO cells restored the anti-proliferative actions of 1,25D. Genomic profiling demonstrated that 1,25D failed to alter gene expression in KO240 cells whereas major changes were observed in WT145 cells and in KO clones stably expressing hVDR (KO(hVDR) cells). With a 2-fold cutoff, 117 transcripts in WT145 cells and 197 transcripts in the KO(hVDR) clones were significantly altered by 1,25D. Thirty-five genes were found to be commonly regulated by 1,25D in all VDR-positive cell lines. Of these, we identified a cohort of four genes (Plau, Hbegf, Postn, Has2) that are known to drive breast cancer invasion and metastasis whose expression was markedly down regulated by 1,25D. These data support a model whereby 1,25D coordinately suppresses multiple proteins that are required for survival of triple-negative/basal-like breast cancer cells. Since studies have demonstrated a high prevalence of vitamin D deficiency in women with basal-like breast cancer, correction of vitamin D deficiency in these women represents a reasonable, but as yet untested, strategy to delay recurrence and extend survival. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Ricciardelli C, Ween MP, Lokman NA, et al.
Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer.
BMC Cancer. 2013; 13:476 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Hyaluronan (HA) an important component of the extracellular matrix, has been linked to tumor progression and drug resistance in several malignancies. However, limited data is available for ovarian cancer. This study investigated the role of hyaluronan (HA) and a potential link between the HA-CD44 pathway and membrane ATP binding cassette (ABC) transporter proteins in ovarian cancer chemoresistance.
METHODS: We investigated the ability of HA to block the cytotoxic effects of the chemotherapy drug carboplatin, and to regulate the expression of ABC transporters in ovarian cancer cells. We also examined HA serum levels in ovarian cancer patients prior to and following chemotherapy and assessed its prognostic relevance.
RESULTS: HA increased the survival of carboplatin treated ovarian cancer cells expressing the HA receptor, CD44 (OVCAR-5 and OV-90). Carboplatin significantly increased expression of HAS2, HAS3 and ABCC2 and HA secretion in ovarian cancer cell conditioned media. Serum HA levels were significantly increased in patients following platinum based chemotherapy and at both 1st and 2nd recurrence when compared with HA levels prior to treatment. High serum HA levels (>50 μg/ml) prior to chemotherapy treatment were associated with significantly reduced progression-free (P = 0.014) and overall survival (P = 0.036). HA production in ovarian cancer cells was increased in cancer tissues collected following chemotherapy treatment and at recurrence. Furthermore HA treatment significantly increased the expression of ABC drug transporters (ABCB3, ABCC1, ABCC2, and ABCC3), but only in ovarian cancer cells expressing CD44. The effects of HA and carboplatin on ABC transporter expression in ovarian cancer cells could be abrogated by HA oligomer treatment. Importantly, HA oligomers increased the sensitivity of chemoresistant SKOV3 cells to carboplatin.
CONCLUSIONS: Our findings indicate that carboplatin chemotherapy induces HA production which can contribute to chemoresistance by regulating ABC transporter expression. The HA-CD44 signaling pathway is therefore a promising target in platinum resistant ovarian cancer.

Niedworok C, Kretschmer I, Röck K, et al.
The impact of the receptor of hyaluronan-mediated motility (RHAMM) on human urothelial transitional cell cancer of the bladder.
PLoS One. 2013; 8(9):e75681 [PubMed] Free Access to Full Article Related Publications
UNLABELLED: Hyaluronan (HA) is a carbohydrate of the extracellular matrix with tumor promoting effects in a variety of cancers. The present study addressed the role of HA matrix for progression and prognosis of human bladder cancer by studying the expression and function of HA-related genes.
METHODS: Tissue samples of 120 patients with different stages of transitional cell bladder cancer, who underwent surgical treatment for bladder cancer at the University Hospital of Essen were analysed. mRNA-expression levels of HA synthases (HAS1-3) and HA-receptors (RHAMM and CD44) were evaluated by real time RT-PCR in comparison to healthy bladder tissue as control. In uni- and multivariate cox proportional hazard survival regression analysis, the impact of the gene expression levels on survival was assessed. In vitro knock-down of RHAMM, CD44 and HAS isoenzymes was achieved by siRNA and lentiviral shRNA in J82 bladder cancer cells. Transfected cells were analysed in vitro with regard to proliferation, cell cycle and apoptosis. J82 cells after knock-down of RHAMM were xenografted into male nu/nu athymic mice to monitor tumor progression in vivo.
RESULTS: In invasive tumor stages RHAMM-, HAS1 and HAS2 mRNA-expression levels were elevated whereas HAS3v1 was reduced as compared to non-invasive tumors. Subsequently, Kaplan-Meier analysis revealed reduced bladder cancer specific survival in patients with high RHAMM mRNA and low HAS3v1 expression. Elevated RHAMM in invasive tumors was confirmed by RHAMM immunohistochemistry. Furthermore, multivariate analysis revealed that only RHAMM expression was associated with poor prognosis independent from other survival factors (HR=2.389, 95% CI 1.227-4.651, p=0.01). Lentiviral RHAMM knock-down revealed reduced J82 cell proliferation in vitro and reduced xenograft tumor growth in vivo.
CONCLUSION: The data suggest that RHAMM plays a crucial role in mediating progression of muscle-invasive bladder cancer and recommends RHAMM for further evaluation as a prognostic marker or therapeutic target in bladder cancer therapy.

Hiraga T, Ito S, Nakamura H
Cancer stem-like cell marker CD44 promotes bone metastases by enhancing tumorigenicity, cell motility, and hyaluronan production.
Cancer Res. 2013; 73(13):4112-22 [PubMed] Related Publications
CD44, an adhesion molecule that binds to the extracellular matrix, primarily to hyaluronan (HA), has been implicated in cancer cell migration, invasion, and metastasis. CD44 has also recently been recognized as a marker for stem cells of several types of cancer. However, the roles of CD44 in the development of bone metastasis are unclear. Here, we addressed this issue by using bone metastatic cancer cell lines, in which CD44 was stably knocked down. Tumor sphere formation and cell migration and invasion were significantly inhibited by CD44 knockdown. Furthermore, the downregulation of CD44 markedly suppressed tumorigenicity and bone metastases in nude mice. Of note, the number of osteoclasts decreased in the bone metastases. Microarray analysis revealed that the expression of HA synthase 2 was downregulated in CD44-knockdown cells. The localization of HA in the bone metastatic tumors was also markedly reduced. We then examined the roles of CD44-HA interaction in bone metastasis using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis. 4-MU decreased tumor sphere and osteoclast-like cell formation in vitro. Moreover, 4-MU inhibited bone metastases in vivo with reduced number of osteoclasts. These results collectively suggest that CD44 expression in cancer cells promotes bone metastases by enhancing tumorigenicity, cell migration and invasion, and HA production. Our results also suggest the possible involvement of CD44-expressing cancer stem cells in the development of bone metastases through interaction with HA. CD44-HA interaction could be a potential target for therapeutic intervention for bone metastases.

Rilla K, Oikari S, Jokela TA, et al.
Hyaluronan synthase 1 (HAS1) requires higher cellular UDP-GlcNAc concentration than HAS2 and HAS3.
J Biol Chem. 2013; 288(8):5973-83 [PubMed] Free Access to Full Article Related Publications
Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.

Kobayashi Y, Banno K, Shimizu T, et al.
Gene expression profile of a newly established choriocarcinoma cell line, iC3-1, compared to existing choriocarcinoma cell lines and normal placenta.
Placenta. 2013; 34(2):110-8 [PubMed] Related Publications
Gestational choriocarcinoma is a malignant trophoblastic tumor that usually occurs in the uterus after pregnancy. The tumor is curable with advanced chemotherapy, but the molecular mechanism of choriocarcinoma tumorigenesis remains unclear. This is partly because the low incidence makes it difficult to obtain clinical samples for investigation and because an appropriate choriocarcinoma cell model to study the tumorigenesis has not been developed. We have established a new choriocarcinoma cell line, induced choriocarcinoma cell-1 (iC(3)-1), that possesses unique characteristics compared to other choriocarcinoma cell lines, including production of tumors that consist of the two types of cells commonly found in choriocarcinoma and mimicking of the clinical pathology. Existing trophoblast cell lines utilized in previous choriocarcinoma studies have had significantly dissimilar gene expression profiles. Therefore, it is important to choose an appropriate cell line for a particular study based on the characteristics of the cell line. In this study, to clarify the genetic characteristics of iC(3)-1 and to explore the tumorigenesis mechanism, we examined the gene profile of iC(3)-1 compared to those of existing cell lines and normal placental tissue. Bioinformatics analysis showed that several characteristic genes, IGF1R, CHFR, MUC3A, TAF7, PARK7, CDC123 and PSMD8, were significantly upregulated in iC(3)-1 compared to BeWo and JEG3 cells. Interestingly, HAS2, CD44 and S100P were significantly upregulated in iC(3)-1 compared to parental HTR8/SVneo cells and normal third trimester placenta. Choriocarcinoma samples also showed immunoreactivity to HAS2, CD44 and S100. In summary, the gene expression profile of iC(3)-1 suggests that studies using this cell line can make an important contribution to improved understanding of choriocarcinoma tumorigenesis.

Guzman L, Adriaenssens T, Ortega-Hrepich C, et al.
Human antral follicles <6 mm: a comparison between in vivo maturation and in vitro maturation in non-hCG primed cycles using cumulus cell gene expression.
Mol Hum Reprod. 2013; 19(1):7-16 [PubMed] Related Publications
Within the context of an oocyte in vitro maturation (IVM) program for reproductive treatment, oocyte cumulus complexes (COCs) derived from follicles <6 mm in patients with PCOS were matured in vitro. Key transcripts related to meiotic maturation (FSHR, LHCGR, EGFR, PGR) and oocyte competence (AREG, ADAMTS, HAS2, PTGS2) were quantified in cumulus cells (CCs) before and after maturation. Control CC samples were collected from PCOS and normo-ovulatory patients who had undergone conventional gonadotrophin stimulation for IVF/ICSI. Additional control samples from a non-stimulated condition were obtained ex vivo from patients undergoing ovariectomy for fertility preservation. Expression data from CCs from follicles with a diameter of <6 mm before (IVM-CCs) and after in vitro maturation (IVM-CCs) were obtained after pooling CCs into four groups in relation to the percentage of matured (MII) oocytes obtained after 40 h of IVM (0; 40-60; 61-80; 100% MII) and values were compared with in vivo matured controls (IVO-CCs). Genes encoding key receptors mediating meiotic resumption are expressed in human antral follicles of <6 mm before and after IVM. The expression levels of FSHR, EGFR and PGR in CCs were significantly down-regulated in the IVO-CCs groups and in the 100% MII IVM group compared with the BM groups; all the receptors studied in the 100% MII IVM group reached an expression profile similar to that of IVO-CCs. However, after maturation in a conventional IVF/ICSI cycle, IVO-CCs from large follicles contained significantly increased levels of ADAMTS1, AREG, HAS2 and PTGS2 compared with IVM-CCs and IVM-CCs; the expression patterns for these genes in all IVM-CCs were unchanged compared with IVM-CCs. In conclusion, genes encoding receptors involved in oocyte meiotic resumption appeared to be expressed in CCs of small human antral follicles. Expression levels of genes-encoding factors reflecting oocyte competence were significantly altered in IVM-CCs compared with in vivo matured oocytes from large follicles. Observed differences might be explained by the different stimulation protocols, doses of gonadotrophin or by the intrinsic differences between in vivo and in vitro maturation.

Kaur S, Archer KJ, Devi MG, et al.
Differential gene expression in granulosa cells from polycystic ovary syndrome patients with and without insulin resistance: identification of susceptibility gene sets through network analysis.
J Clin Endocrinol Metab. 2012; 97(10):E2016-21 [PubMed] Free Access to Full Article Related Publications
CONTEXT: Polycystic ovary syndrome (PCOS) is a heterogeneous, genetically complex, endocrine disorder of uncertain etiology in women.
OBJECTIVE: Our aim was to compare the gene expression profiles in stimulated granulosa cells of PCOS women with and without insulin resistance vs. matched controls.
RESEARCH DESIGN AND METHODS: This study included 12 normal ovulatory women (controls), 12 women with PCOS without evidence for insulin resistance (PCOS non-IR), and 16 women with insulin resistance (PCOS-IR) undergoing in vitro fertilization. Granulosa cell gene expression profiling was accomplished using Affymetrix Human Genome-U133 arrays. Differentially expressed genes were classified according to gene ontology using ingenuity pathway analysis tools. Microarray results for selected genes were confirmed by real-time quantitative PCR.
RESULTS: A total of 211 genes were differentially expressed in PCOS non-IR and PCOS-IR granulosa cells (fold change≥1.5; P≤0.001) vs. matched controls. Diabetes mellitus and inflammation genes were significantly increased in PCOS-IR patients. Real-time quantitative PCR confirmed higher expression of NCF2 (2.13-fold), TCF7L2 (1.92-fold), and SERPINA1 (5.35-fold). Increased expression of inflammation genes ITGAX (3.68-fold) and TAB2 (1.86-fold) was confirmed in PCOS non-IR. Different cardiometabolic disease genes were differentially expressed in the two groups. Decreased expression of CAV1 (-3.58-fold) in PCOS non-IR and SPARC (-1.88-fold) in PCOS-IR was confirmed. Differential expression of genes involved in TGF-β signaling (IGF2R, increased; and HAS2, decreased), and oxidative stress (TXNIP, increased) was confirmed in both groups.
CONCLUSIONS: Microarray analysis demonstrated differential expression of genes linked to diabetes mellitus, inflammation, cardiovascular diseases, and infertility in the granulosa cells of PCOS women with and without insulin resistance. Because these dysregulated genes are also involved in oxidative stress, lipid metabolism, and insulin signaling, we hypothesize that these genes may be involved in follicular growth arrest and metabolic disorders associated with the different phenotypes of PCOS.

Yang X, Rutnam ZJ, Jiao C, et al.
An anti-let-7 sponge decoys and decays endogenous let-7 functions.
Cell Cycle. 2012; 11(16):3097-108 [PubMed] Free Access to Full Article Related Publications
The let-7 family contains 12 members, which share identical seed regions, suggesting that they may target the same mRNAs. It is essential to develop a means that can regulate the functions of all members. Using a DNA synthesis technique, we have generated an anti-let-7 sponge aiming to modulate the function of all members. We found that products of the anti-let-7 construct could bind and inactivate all members of the let-7 family, producing decoy and decay effects. To test the role of the anti-let-7 sponge, we stably expressed the anti-let-7 construct in two types of cells, the breast carcinoma cells MT-1 and the oldest and most commonly used human cervical cancer cell line, HeLa cells. We found that expression of anti-let-7 increased cell survival, invasion and adhesion, which corroborate with known functions of let-7 family members. We further identified a novel target site across all species of the let-7 family in hyaluronan synthase 2 (HAS2). HAS2 overexpression produced similar effects as the anti-let-7 sponge. Silencing HAS2 expression by siRNAs produced opposite effects to anti-let-7 on cell survival and invasion. The ability of anti-let-7 to regulate multiple members of the let-7 family allows us to observe their multiple functions using a single reagent. This approach can be applied to other family members with conserved sequences.

Tamada Y, Takeuchi H, Suzuki N, et al.
Cell surface expression of hyaluronan on human ovarian cancer cells inversely correlates with their adhesion to peritoneal mesothelial cells.
Tumour Biol. 2012; 33(4):1215-22 [PubMed] Related Publications
Eight of 15 human ovarian carcinoma cell lines were shown to express high levels of hyaluronan (HA) on their surfaces. The role of cell surface HA in its adhesion to mesothelial cells, which is potentially involved in peritoneal dissemination, was evaluated. Three human ovarian carcinoma cell lines, ES-2, MH, and KF cells, were repeatedly sorted into variant cell lines with high levels of cell surface HA (ES-2/HA+7, MH/HA+7, and KF/HA+7) and with low cell surface HA (ES-2/HA-7, MH/HA-7, and KF/HA-7). The ability of these cells to adhere to peritoneal mesothelial cells was compared. ES-2/HA+7, MH/HA+7, and KF/HA+7 cells were less adherent to mesothelial cells than the ES-2/HA-7, MH/HA-7, and KF/HA-7 cells. On ovarian carcinoma cells, high cell surface HA levels seem to inversely correlate with their capacity to adhere and disseminate to the peritoneum. Considering that peritoneum implantation is the primary ovarian cancer complication, HA cell surface expression may be considered a property associated with a less aggressive phenotype, which is contrary to the general perception that HA expression is associated with malignant progression.

Wan X, Liu J, Lu JF, et al.
Activation of β-catenin signaling in androgen receptor-negative prostate cancer cells.
Clin Cancer Res. 2012; 18(3):726-36 [PubMed] Free Access to Full Article Related Publications
PURPOSE: To study Wnt/β-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the β-catenin-androgen receptor (AR) interaction.
EXPERIMENTAL DESIGN: We carried out β-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of β-catenin-mediated transcription), and sequenced the β-catenin gene in MDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down β-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed β-catenin and AR in 27 bone metastases of human CRPCs.
RESULTS: β-Catenin nuclear accumulation and TOP-flash reporter activity were high in MDA prostate cancer 118b but not in MDA prostate cancer 2b or PC-3 cells. MDA prostate cancer 118a and MDA prostate cancer 118b cells carry a mutated β-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated β-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant β-catenin. Finally, we found nuclear localization of β-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher's exact test), suggesting that reduced AR expression enables Wnt/β-catenin signaling.
CONCLUSION: We identified a previously unknown downstream target of β-catenin, HAS2, in prostate cancer, and found that high β-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone metastatic prostate cancer. These findings may guide physicians in managing these patients.

Okuda H, Kobayashi A, Xia B, et al.
Hyaluronan synthase HAS2 promotes tumor progression in bone by stimulating the interaction of breast cancer stem-like cells with macrophages and stromal cells.
Cancer Res. 2012; 72(2):537-47 [PubMed] Free Access to Full Article Related Publications
The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here, we report that upregulation of hyaluronan synthase 2 (HAS2) occurs in highly metastatic breast cancer stem-like cells (CSC) defined by CD44(+)/CD24(-)/ESA(+) phenotype, where it plays a critical role in the generation of a prometastatic microenvironment in breast cancer. HAS2 was critical for the interaction of CSCs with tumor-associated macrophages (TAM), leading to enhanced secretion of platelet-derived growth factor-BB from TAMs, which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone, an inhibitor of HAS, which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings show a critical role of HAS2 in the development of a prometastatic microenvironment and suggest that HAS2 inhibitors can act as antimetastatic agents that disrupt a paracrine growth factor loop within this microenvironment.

Bernert B, Porsch H, Heldin P
Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1).
J Biol Chem. 2011; 286(49):42349-59 [PubMed] Free Access to Full Article Related Publications
Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.

Urakawa H, Nishida Y, Knudson W, et al.
Therapeutic potential of hyaluronan oligosaccharides for bone metastasis of breast cancer.
J Orthop Res. 2012; 30(4):662-72 [PubMed] Related Publications
Hyaluronan (HA) oligosaccharides were reported to have suppressive effects on various malignant tumors via disruption of receptor HA interactions. However, no studies have focused on the effects of HA oligosaccharides on bone metastasis of breast cancer. In this study, we clarified the effective size of HA oligosaccharides required to inhibit cell growth in the highly invasive breast cancer cell line, MDA-MB-231 cells. Based on the results of cell growth assay, we subsequently analyzed the effects of HA tetrasaccharides, HA decasaccharides, and high molecular weight HA on the other breast cancer cell behaviors in vitro and breast cancer bone metastasis in vivo. HA decasaccharides significantly inhibited cell growth, motility, and invasion, whereas tetrasaccharides did not. HAS2 mRNA expression was altered after the treatment with both tetrasaccharides and decasaccharides. Phosphorylation of Akt was suppressed after 1 h treatment with HA decasaccharides, and the effect was partially reversed by anti-CD44 monoclonal antibody. In vivo, local application of HA decasaccharides inhibited the expansion of osteolytic lesions in tibia on soft X-rays using mouse bone metastasis model of breast cancer. Histological analysis revealed HA accumulation in bone metastatic lesions was perturbed by decasaccharides. These results suggest that HA oligosaccharides suppressed progression of bone metastasis in breast cancer via interruption of endogenous HA-CD44 interaction, and as such, can be a novel therapeutic candidate to limit bone metastasis of breast cancer.

Chi A, Shirodkar SP, Escudero DO, et al.
Molecular characterization of kidney cancer: association of hyaluronic acid family with histological subtypes and metastasis.
Cancer. 2012; 118(9):2394-402 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Molecular profiling of renal cell carcinomas (RCCs) may improve the distinction between oncocytoma and malignant RCC subtypes and aid in early detection of metastasis. The hyaluronic acid (HA) family includes HA synthases (HAS1, HAS2, HAS3), hyaluronidases (HYAL-1, HYAL-2, HYAL-3, HYAL-4, PH20, HYAL-P1), and HA receptors (CD44s, CD44v, RHAMM). HA family members promote tumor growth and metastasis. The authors evaluated the expression of HA family members in kidney specimens.
METHODS: By using quantitative polymerase chain reaction, mRNA levels of 12 HA family members were measured in tumor specimens obtained from 86 consecutive patients undergoing nephrectomy; 80 of them also provided normal specimens. Mean and median follow-up were 15.2 ± 8.8 and 13.8 months. RCC specimens included clear cell RCC: 65; papillary: 10; chromophobe: 5; oncocytoma: 6; metastasis positive: 17.
RESULTS: Median HAS1, CD44s, and RHAMM transcript levels were elevated 3- to 25-fold in clear cell RCC and papillary and chromophobe tumors when compared with normal tissues. HYAL-4, CD44s, and RHAMM levels were elevated 4- to 12-fold in clear cell RCC and papillary tumors when compared with oncocytomas; only HYAL-4 levels distinguished between chromophobe and oncocytoma (P = .009). CD44s and RHAMM levels were significantly higher in tumors <4 cm (510 ± 611 and 19.6 ± 20.8, respectively) when compared with oncocytoma (46.4 ± 20 and 3.8 ± 2.5; P ≤ .006). In univariate and multivariate analyses, CD44s (P < .0001), RHAMM (P < .0001), stage, tumor size, and/or renal vein involvement were significantly associated with metastasis. The combined CD44s + RHAMM marker had 82% sensitivity and 86% specificity to predict metastasis.
CONCLUSIONS: CD44s and RHAMM levels distinguish between oncocytoma and RCC subtypes regardless of tumor size and are potential predictors of RCC metastasis.

Kramer MW, Escudero DO, Lokeshwar SD, et al.
Association of hyaluronic acid family members (HAS1, HAS2, and HYAL-1) with bladder cancer diagnosis and prognosis.
Cancer. 2011; 117(6):1197-209 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Cancer biomarkers are the backbone for the implementation of individualized approaches to bladder cancer (BCa). Hyaluronic acid (HA) and all 7 members of the HA family, that is, HA synthases (HA1, HA2, HA3), HYAL-1 hyaluronidase, and HA receptors (CD44s, CD44v, and RHAMM), function in tumor growth and progression. However, the diagnostic and prognostic potential of these 7 HA family members has not been compared simultaneously in any cancer. We evaluated the diagnostic and prognostic potential of HA family members in BCa.
METHODS: Using quantitative PCR and immunohistochemistry, expression of HA family members was evaluated in prospectively collected bladder tissues (n = 72); mean and median follow-up were 29.6 ± 5.3 and 24 months, respectively. Transcript levels were also measured in exfoliated urothelial cells from urine specimens (n = 148).
RESULTS: Among the HA family members, transcript levels of the HA synthases, HYAL-1, CD44v, and RHAMM were 4- to 16-fold higher in BCa tissues than in normal tissues (P < .0001); however, CD44s levels were lower. In univariate and multivariate analyses, tumor stage (P = .003), lymph node invasion (P = .033), HYAL-1 (P = .019), and HAS1 (P = .027) transcript levels, and HYAL-1 staining (P = .021) were independently associated with metastasis. Tumor stage (P = .019) and HYAL-1 (P = .046) transcript levels were also associated with disease-specific mortality. Although HA synthase and HYAL-1 transcript levels were elevated in exfoliated urothelial cells from BCa patients, the combined HAS2-HYAL-1 expression detected BCa with an overall sensitivity of 85.4% and a specificity of 79.5% and predicted BCa recurrence within 6 months (P = .004; RR = 6.7).
CONCLUSIONS: HYAL-1 and HAS1 expression predicted BCa metastasis, and HYAL-1 expression also predicted disease-specific survival. Furthermore, the combined HAS2-HYAL-1 biomarker detected BCa and significantly predicted its recurrence.

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