Development and validation of H11B2C2 monoclonal antibody-reactive hyaluronic acid binding protein: overexpression of HABP during human tumor progression.

Informative biomarkers of tumor progression have been elusive. The interaction between hyaluronic acid (HA) and its binding proteins (HABP) plays a pivotal role during malignancy. In the present study, we have developed a monoclonal antibody (mAb, termed as H11B2C2 mAb) and showed that this mAb specifically reacts with overexpressed HABP from a wide variety of malignant tumors as compared with benign tumors. In Western blot analysis, H11B2C2 mAb detected a major 80-kDa protein from human cancer cell lines, and the overexpression of 55-57- and 30-kDa proteins in malignant tumors compared with benign tumors. Furthermore, immunohistochemical analysis of different types of benign and malignant tumors with different grades showed higher expression of HABP in all the malignant tumors when compared with the benign tumors. HABP overexpression was specific to tumor cells when compared with the surrounding stroma and localized on the cell surface as well as in the intracellular region. The competitive inhibition experiments using HA polymer and its oligosaccharides in the Western blot and immunohistopathology experiments suggested that the H11B2C2 mAb-reactive protein is HABP. Altogether, the present study showed overexpression of the H11B2C2 mAb-reactive HABP in various malignant tumors as compared with benign tumors. Thus, H11B2C2 mAb-reactive HABP can be used as a potential biomarker during tumor progression.

Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients.

INTRODUCTION: Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease. METHODS: Sera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21 and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III) and the small nucleolar RNU44 (SNORD44), were also analyzed for normalization. Significant results from the initial study were verified using a second set of sera from 15 healthy patients, 15 breast cancer patients without clinical disease and 15 with metastatic disease, and a third set of 12 healthy and 18 patients with metastatic disease. U6 was further verified in the extended second cohort of 75 healthy and 68 breast cancer patients without clinical disease. RESULTS: U6:SNORD44 ratio was consistently higher in breast cancer patients with or without active disease (fold change range 1.5-6.6, p value range 0.0003 to 0.05). This increase in U6:SNORD44 ratio was observed in the sera of both estrogen receptor-positive (ER+) and ER-negative breast cancer patients. MiR-16 and 5S, which are often used as normalization controls for microRNAs, showed remarkable experimental variability and thus are not ideal for normalization. CONCLUSIONS: Elevated serum U6 levels in breast cancer patients irrespective of disease activity at the time of serum collection suggest a new paradigm in cancer; persistent systemic changes during cancer progression, which result in elevated activity of RNAP-III and/or the stability/release pathways of U6 in non-cancer tissues. Additionally, these results highlight the need for developing standards for normalization between samples in microRNA-related studies for healthy versus cancer and for inter-laboratory reproducibility. Our studies rule out the utility of miR-16, U6 and 5S RNAs for this purpose.

Expression of hyaluronan in human tumor progression.

BACKGROUND: The development and progression of human tumors is accompanied by various cellular, biochemical and genetic alterations. These events include tumor cells interaction with extracellular matrix molecules including hyaluronan (HA). Hyaluronan is a large polysaccharide associated with pericellular matrix of proliferating, migrating cells. Its implication in malignant transformation, tumor progression and with the degree of differentiation in various invasive tumors has well accepted. It has been well known the role HA receptors in tumor growth and metastasis in various cancer tissues. Previously we have observed the unified over expression of Hyaluronic Acid Binding Protein (HABP), H11B2C2 antigen by the tumor cells in various types progressing tumor tissues with different grades. However, the poor understanding of relation between HA and HA-binding protein expression on tumor cells during tumor progression as well as the asymmetric observations of the role of HA expression in tumor progression prompted us to examine the degree of HA expression on tumor cells vs. stroma in various types of human tumors with different grades. METHODS: In the present study clinically diagnosed tumor tissue samples of different grades were used to screen the histopathological expression of hyaluronan by using b-PG (biotinylated proteoglycan) as a probe and we compared the relative HA expression on tumor cells vs. stroma in well differentiated and poorly differentiated tumors. Specificity of the reaction was confirmed either by pre-digesting the tissue sections with hyaluronidase enzyme or by staining the sections with pre-absorbed complex of the probe and HA-oligomers. RESULTS: We show here the down regulation of HA expression in tumor cells is associated with progression of tumor from well differentiated through poorly differentiated stage, despite the constant HA expression in the tumor associated stroma. CONCLUSION: The present finding enlighten the relative roles of HA expression on tumor vs. stroma during the progression of tumors.