Receptor For Hyaluronic Acid-mediated Motility Expression Research Articles

RHAMM/hyaluronan inhibit β-catenin degradation, enhance downstream signaling, and facilitate fibrosarcoma cell growth.

Increased hyaluronan deposition (HA) in various cancer tissues, including sarcomas, correlates with disease progression. The receptor for hyaluronic acid-mediated motility (RHAMM) expression is elevated in most human cancers. β-catenin is a critical downstream mediator of the Wnt signaling pathways, facilitating carcinogenic events characterized by deregulated cell proliferation. We previously showed that low molecular weight (LMW) HA/RHAMM/β-catenin signaling axis increases HT1080 fibrosarcoma cell growth. Here, focusing on mechanistic aspects and utilizing immunofluorescence and immunoprecipitation, we demonstrate that LMW HA treatment enhanced RHAMM intracellular localization (p ≤ 0.001) and RHAMM/β-catenin colocalization in HT1080 fibrosarcoma cells (p ≤ 0.05). Downregulating endogenous HA attenuated the association of RHAMM/β-catenin in HT1080 fibrosarcoma cells (p ≤ 0.0.01). Notably, Axin-2, the key β-catenin degradation complex component, and RHAMM were demonstrated to form a complex primarily to cell membranes, enhanced by LMW HA (p ≤ 0.01). In contrast, LMW HA attenuated the association of β-catenin and Axin-2 (p ≤ 0.05). The utilization of FH535, a Wnt signaling inhibitor, showed that LMW HA partially rescued the Wnt-dependent growth of HT1080 cells and restored the expression of Wnt/β-catenin mediators, cyclin-D1 and c-myc (p ≤ 0.05). B6FS fibrosarcoma cells with different HA metabolism do not respond to the LMW HA growth stimulus (p = NS). The present study identifies a novel LMW HA/RHAMM mechanism in a fibrosarcoma model. LMW HA regulates intracellular RHAMM expression, which acts as a scaffold protein binding β-catenin and Axin-2 at different cellular compartments to increase β-catenin expression, transcriptional activity, and fibrosarcoma growth.

Receptor for Hyaluronic Acid-mediated Motility (RHAMM) Is Associated With Prostate Cancer Migration and Poor Prognosis.

Hyaluronic acid (HA) is a large glycosaminoglycan composed of an extracellular matrix. The HA-rich microenvironment and receptors of HA have been suggested to play roles in cancer progression. The biological and clinical significance of receptor for HA-mediated motility (RHAMM), known as CD168 in prostate cancer (PC) remains unknown. This study aimed to investigate the expression of RHAMM, as well as its functional and clinical relevance in PC. HA concentration and RHAMM mRNA expression were examined in 3 PC cell lines (LNCaP, PC3 and DU145). We investigated the effect of HA and RHAMM on the migratory ability of PC cells using a transwell migration assay. Immunohistochemistry was also used to evaluate the RHAMM expression pattern in pre-treatment tissue samples from 99 patients with metastatic hormone-sensitive PC (HSPC) who received androgen deprivation therapy (ADT). HA was secreted in all cultured PC cell lines. Among the total HA, low-molecular-weight HA (LMW-HA) (<100 kDa) was detected all examined cell lines. The number of migration cells was significantly increased by adding LMW-HA. RHAMM mRNA expression was increased in DU145 cells. Knockdown of RHAMM using small-interfering RNA resulted in decreased cell migration. Immunohistochemical analysis revealed strong RHAMM expression in 31 (31.3%) patients with metastatic HSPC. A strong RHAMM expression was significantly associated with short ADT duration and poor survival in univariate and multivariate analyses. The size of HA is important in terms of PC progression. LMW-HA and RHAMM enhanced PC cell migration. RHAMM could be used as a novel prognostic marker in patients with metastatic HSPC.

Organic ultraviolet filters regulate hyaluronan metabolism in human epidermal keratinocytes through the phosphatidylinositol 3-kinase pathway

Chronic exposure of skin to ultraviolet (UV) radiation is responsible for skin ageing, which includes degradation of the epidermal and dermal layers. Filtering UV light is key in the sunscreen industry. We studied the effects of organic UV filters on hyaluronan (HA) metabolism and skin hydration in human HaCaT keratinocytes. The gene expression of HA receptors, HA synthase (HAS), hyaluronidase (HYAL), and water channel aquaporin 3 (AQP3) was evaluated by quantitative RT–PCR. The state of oxidative stress was determined by measuring the intracellular levels of reactive oxygen species (ROS). The results showed that five organic UV filters reduced the extracellular contents of HA, and a phosphatidylinositol 3-kinase (PI3K) inhibitor partially restored the decreased HA levels after octinoxate, octocrylene, and oxybenzone treatment. The expression levels of HA receptors, including cluster of differentiation 44 (CD44), receptor for hyaluronic acid-mediated motility (RHAMM), and toll-like receptors (TLRs), were determined. Avobenzone, octinoxate, oxybenzone, and padimate O exerted inhibitory effects on RHAMM expression. Oxybenzone led to a significant increase in CD44 and AQP3 expression. Both octinoxate and octocrylene increased TLR4 expression but decreased ROS accumulation by activating the PI3K pathway. However, the organic UV filters differentially regulated the mRNA expression of HAS and HYAL. Taken together, these results suggest that certain organic UV filters regulate HA metabolism in human keratinocytes in a PI3K pathway-dependent manner.

Abstract 5990: Targeted nanoparticles reduce tumor progression and lung metastasis by limiting DNA damage repair and immune suppression in metastatic triple-negative breast cancer

Abstract Background: Metastatic triple-negative breast cancer (TNBC), especially with BRCA1/2 mutations, is a deadly subtype of breast cancer with ~12% of 5-year survival. Despite their FDA approval, the therapeutic benefits of poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPi) are limited to BRCA1/2 mutated malignancies. Moreover, the PARPi olaparib has been shown to upregulate the expression of programmed death-ligand 1 (PD-L1) leading to immune suppression. The purpose of this study is to investigate if a novel nanoparticle formulation of oligomer hyaluronic acid (oHA) and doxorubicin (DOX) co-loaded in an iRGD-conjugated polymer-lipid nanocarrier (iRGD-DOX-oHA-PLN) can inhibit DNA damage repair and suppress PD-L1 in both mutant and non-mutant TNBC cells and reduce tumor progression and lung metastasis compared to the PARPi olaparib. We postulate that the co-administered oHA will block the signaling pathways of native hyaluronic acid (HA) receptors, the cluster of differentiation 44 (CD 44) and receptor for HA mediated motility (RHAMM), that regulate DNA damage repair and immunosuppression and enhance DOX efficacy. Methods: In vitro cellular uptake of various DOX formulations by TNBC MDA-MB-231-luc-D3H2LN cells and MDA-MB-436 (BRCA1 mutant) cells was examined using confocal laser scanning microscopy or spectrophotometer. The expression level of RHAMM, PD-L1, and PARP1 parylation (PAR) was evaluated after treatment with iRGD-DOX-oHA-PLN or olaparib in both cell lines using western blot or confocal laser scanning microscopy. The therapeutic efficacy of iRGD-DOX-oHA-PLN compared to olaparib was determined by monitoring the tumor progression and lung metastasis development in orthotopic breast tumor models of MDA-MB-231-luc-D3H2LN or MDA-MB-436 cell line. Results: The expression of native HA receptor RHAMM was profoundly reduced by ~5 folds in vivo and the DNA DSB was significantly increased by the iRGD-DOX-oHA-PLN treatment with 40% of γH2AX positive cells in tumor tissue sections. The iRGD-functionalized PLN enhanced DOX cellular uptake compared to DOX free drug or non-targeted NPs by ~3- and ~1.5-fold, in MDA-MB-231 and MDA-MB-436 cells, respectively. While olaparib upregulated PD-L1 and free DOX increased PAR level, the iRGD-DOX-oHA-PLN reduced the parylation and PD-L1 expression in both TNBC cell lines. The iRGD-DOX-oHA-PLN treatment (two dose of 10 mg/kg i.v. biweekly) outperformed olaparib (twenty doses of 50 mg/kg i.p 5x/week) in preventing tumor progression and lung metastasis in vivo over a 4-week period. Conclusion: The results suggest that the iRGD-DOX-oHA-PLN can effectively inhibit DNA damage repair and immunosuppression of cancer cells and could be a promising multitargeted nanomedicine for the treatment of both BRCA1-mutant and non-mutant metastatic TNBC. Citation Format: Ibrahim Alradwan, Pei Zhi, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Rauth, Xiao Yu Wu. Targeted nanoparticles reduce tumor progression and lung metastasis by limiting DNA damage repair and immune suppression in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5990.

Co-localization and crosstalk between CD44 and RHAMM depend on hyaluronan presentation

CD44 and the receptor for hyaluronic acid-mediated motility (RHAMM) are the main hyaluronan (HA) receptors. They are commonly overexpressed in different cancers activating signaling pathways related to tumor progression, metastasis and chemoresistance. Besides their involvement in signal transduction via interaction with HA, currently, there is a little information about the possible crosstalk between CD44 and RHAMM and the role of HA in this process. In the present work, we used immunocytochemistry combined with Förster resonance energy transfer (FRET) microscopy and co-immunoprecipitation to elucidate the involvement of HA in CD44 and RHAMM expression, co-localization and crosstalk. We studied breast cancer cells lines with different degrees of invasiveness and expression of these receptors in the absence of exogenous HA and compared the data with the results obtained for cultures supplemented with either soluble HA or seeded on substrates with end-on immobilized HA. Our results demonstrated that cells response depends on the HA presentation: CD44/RHAMM complexation was upregulated in all cell lines upon interaction with immobilized HA, but not with its soluble form. Moreover, the results showed that the expression of both CD44 and RHAMM is regulated via interactions with HA indicating cell-specific feedback loop(s) in the signaling cascade.

Expression of the Receptor for Hyaluronic Acid-Mediated Motility (RHAMM) in Endometrial Cancer is Associated With Adverse Histologic Parameters and Tumor Progression.

Endometrial cancer is one of the most common gynecologic malignancies worldwide. Only 2 agents have been approved by Food and Drug Administration for endometrial cancer since 1971. There is a need to identify molecular targets to treat advanced endometrial cancer. The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various types of cancer. Here, we aimed to determine the clinical significance of RHAMM expression in endometrial cancer. Two hundred twenty-five cases of endometrial cancer, including serous and endometrioid types, and 8 cases of normal endometrium were used for studying RHAMM protein levels. The Cancer Genome Atlas database was also queried for RHAMM mRNA expression in endometrial cancer. Increased expression of RHAMM protein was seen in endometrial cancer compared with no or weak expression in normal endometrium. RHAMM expression positively correlated with tumor grade. RHAMM expression was significantly increased in endometrial serous carcinomas, which are high-grade, aggressive types of endometrial cancer, compared with the relatively less aggressive endometrioid carcinomas. RHAMM expression also correlated with the presence of lymphovascular invasion. RHAMM mRNA expression correlated with decreased survival in The Cancer Genome Atlas cohort. Therefore, increased RHAMM expression in endometrial cancer is associated with high-grade tumors and is indicative of more aggressive behavior. These findings suggest RHAMM as a prognostic factor in endometrial cancer and as a potential therapeutic target in advanced endometrial cancer for future studies.

Immunohistochemical analysis of RHAMM expression in normal and neoplastic human tissues: a cell cycle protein with distinctive expression in mitotic cells and testicular germ cells.

Expression of Receptor for Hyaluronic Acid Mediated Motility (RHAMM) increases cellular motility and RHAMM overexpression promotes invasive phenotype and metastasis of cancer cells. RHAMM has been suggested as a biomarker for poor prognosis in several tumor types, including lung, breast, colorectal, gastric, pancreatic ductal, and ovarian cancers. RNA studies showed restricted RHAMM expression in normal tissues, but its protein expression data in tissues were limited. In light of its potential as a prognostic marker and a therapeutic target, we performed immunohistochemical analysis to systematically characterize RHAMM expression in normal and neoplastic human tissues. Among 29 normal adult tissues, RHAMM protein showed restricted expression and was observed in the thymus, lymph node/tonsil, small intestine, colon, skin, bone marrow, placenta, and testis. The cellular distribution patterns of RHAMM in these normal tissues were consistent with RHAMM being a G2/M cell cycle protein, and this was further supported in comparison to the expression of cyclin B2, another G2/M protein. However, unlike the subcellular localization of cyclin B2, RHAMM decorated mitotic spindles in both anaphase and metaphase. RHAMM expression in tumor tissues is variable; and higher RHAMM protein expression is associated with histologically higher-grade tumors in general. Distinct from its expression in somatic tissues, RHAMM showed diffuse, strong, stage-specific expression in the spermatocyte stage of germ cells in adult testis. The neoplastic counterpart, spermatocytic tumor, also showed strong RHAMM expression. This unique expression in testis suggests that RHAMM may function during normal testicular germ cell maturation.

Hyaluronic acid enhances cell migration and invasion via the YAP1/TAZ-RHAMM axis in malignant pleural mesothelioma.

Most malignant mesotheliomas (MPMs) frequently show activated forms of Yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ), which transcriptionally regulates the receptor for hyaluronic acid-mediated motility (RHAMM). As RHAMM is involved in cell migration and invasion in various tumors, we speculated that hyaluronic acid (HA) in pleural fluid might affect the progression of mesothelioma by stimulating cell migration and invasion through RHAMM. The level of RHAMM expression was decreased by YAP1/TAZ knockdown, and conversely increased by forced expression of the active form of YAP1, suggesting that RHAMM was regulated by YAP1/TAZ in MPM cells. Cell migration and invasion were also decreased by YAP1/TAZ or RHAMM knockdown. Notably, HA treatment increased cell motility and invasion, and this was abolished by RHAMM knockdown, suggesting that HA may augment local progression of MPM cells via RHAMM. Furthermore, treatment with fluvastatin, which regulates RHAMM transcription by modulating YAP1/TAZ activity, decreased the motility and invasion of MPM cells. Collectively, these data suggest that HA is an “unfavorable” factor because it promotes malignancy in mesothelioma and that the YAP1/TAZ-RHAMM axis may have potential value as a therapeutic target for inhibition of disease progression in MPM.

Expression of the receptor for hyaluronic acid mediated motility (RHAMM) is associated with poor prognosis and metastasis in non-small cell lung carcinoma.

The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various cancers, but its role in primary and metastatic non-small cell lung carcinoma (NSCLC) remains to be determined. Here, we investigate the clinical relevance of RHAMM expression in NSCLC. RHAMM protein expression correlates with histological differentiation stages and extent of the primary tumor (T stages) in 156 patients with primary NSCLC. Importantly, while focal RHAMM staining pattern is present in 57% of primary NSCLC, intense RHAMM protein expression is present in 96% of metastatic NSCLC cases. In a publicly available database, The Cancer Genome Atlas (TCGA), RHAMM mRNA expression is 12- and 10-fold higher in lung adenocarcinoma and squamous lung carcinoma than in matched normal lung tissues, respectively. RHAMM mRNA expression correlates with stages of differentiation and inferior survival in more than 400 cases of lung adenocarcinoma in the Director's Challenge cohort. Of 4 RHAMM splice variants, RHAMMv3 (also known as RHAMMB) is the dominant variant in NSCLC. Moreover, shRNA-mediated knockdown of RHAMM reduced the migratory ability of two lung adenocarcinoma cell lines, H1975 and H3255. Taken together, RHAMM, most likely RHAMMv3 (RHAMMB), can serve as a prognostic factor for lung adenocarcinomas and a potential therapeutic target in NSCLC to inhibit tumor migration.

Receptor for hyaluronic acid- mediated motility (RHAMM) regulates HT1080 fibrosarcoma cell proliferation via a β-catenin/c-myc signaling axis

BackgroundHigh levels of hyaluronan (HA) synthesis in various cancer tissues, including sarcomas, are correlated with tumorigenesis and malignant transformation. RHAMM (receptor for hyaluronic acid-mediated motility) is overexpressed during tumor development in different malignancies. β-Catenin is a crucial downstream mediator of the Wnt signaling cascade which facilitates carcinogenic events characterized by deregulated cell proliferation. MethodsReal-time PCR, in vitro cell proliferation assay, siRNA transfection, flow cytometry, immunoprecipitation, western blotting and immunofluorescence were utilized. ResultsThe reduction of RHAMM expression was strongly correlated with an inhibition of HT1080 fibrosarcoma cell growth (p≤0.01). LMWHA, in a RHAMM-dependent manner increases cell growth of HT1080 cells ((p≤0.01). Both basal and LMWHA dependent growth of HT1080 cells was attenuated by β-catenin deficiency (p≤0.01). β-Catenin cytoplasmatic deposition is positively regulated by RHAMM (p≤0.01). Immunoflourescence and immunoprecipitation suggest that RHAMM/β-catenin form an intracellular complex. Transfection experiments identified c-myc as candidate downstream mediator of RHAMM/β-catenin effects on HT1080 fibrosarcoma cell proliferation. ConclusionsLMWHA/RHAMM downstream signaling regulates fibrosarcoma cell growth in a β-catenin/c-myc dependent manner. General significanceThe present study suggests that RHAMM is a novel β-catenin intracellular binding partner, protecting β-catenin from degradation and supporting the nuclear translocation of this key cellular mediator, which results in c-myc activation and enhanced fibrosarcoma cell growth.

Receptor for hyaluronic acid-mediated motility (RHAMM, CD168) expression is prognostically important in both nodal negative and nodal positive large cell lung cancer

ObjectiveDespite advances in therapy, lung cancer is still the leading cause of cancer-related mortality in the world. Further prognostic tools are warranted for risk-adapted therapeutic decisions. We analysed a cohort...

Induction of Posterior Capsule Opacification by Hyaluronic Acid in an Ex Vivo Model

Because hyaluronic acid (HA) is found in many surgical viscoelastic agents, this study aimed to determine (1) if HA receptors are present in the canine lens, (2) if the rate of lens epithelial cell (LEC) migration is altered following treatment with HA, and (3) if introduction of exogenous HA into the lens capsule promotes lenticular migration, thus contributing to posterior capsule opacification (PCO). Normal and cataractous canine LECs were evaluated for expression of the HA receptor CD44 and the receptor for HA mediated motility (RHAMM) using immunohistochemistry, immunoblotting, and real-time PCR. Canine LEC were treated with various concentrations of HA, and induction of migration was monitored over time. Commercially available surgical viscoelastics were utilized ex vivo, and rates of PCO formation were analyzed. Basal protein and mRNA expression of both CD44 and RHAMM was noted. Cataractous canine LEC demonstrated significantly (P < 0.01) higher expression of CD44 but not RHAMM. Treatment with higher concentrations of HA resulted in a significant (P < 0.01) increase in CD44 mRNA and increased LEC migration in vitro. Use of CD44-neutralizing antibodies confirmed the role of CD44 in HA-induced lenticular migration. Viscoelastic material containing higher concentrations of HA led to increased rates of ex vivo PCO. Exogenous HA can induce lenticular migration and CD44 expression. Use of surgical viscoelastics that contained HA resulted in increased rates of ex vivo PCO suggesting that judicious selection and use of viscoelastic material during cataract surgery is warranted.

Identification of RHAMM-Derived CD8+ Restricted, Heteroclitical, Cryptic Epitope R9Y as a Promising Target for Immunotherapy of Chronic Lymphocytic Leukemia.

Abstract 3034Poster Board II-1010The receptor for hyaluronic acid mediated motility (RHAMM) is leukemia associated antigen (LAA) strongly correlated with proliferation and bearing negative prognosis for chronic lymphocytic leukemia (CLL). Recently, we completed a peptide vaccination clinical trial with dominant RHAMM-derived epitope (R3) for patients with CLL. To our surprise, R3 vaccination induced regulatory T cells (Treg) in 66% vaccinated patients which might limit the clinical efficacy of peptide immunotherapy. Obtained results also pointed to a pre-existing tolerance to LAA-derived peptides. While the tolerance is limited to dominant epitopes we aimed to identify a crypitic epitopes that might circumvent tolerance mechanisms and in order to increase affinity modified them heteroclitically.We screened in silico 50 peptides with different heteroclitical modifications using SYFPEITHI algorithm and calculate affinity to MHC-I complex. Further, we choose 10 peptide pairs (cryptic – modified) which affinity increased to the highest extent and screened them in vitro assay for the affinity as well as the peptide-MHC-I complex stability in T2 binding test. In functional studies we screened 5 peptide pairs by ELISPOT analysis using CD8+ T cells isolated from peripheral blood (PB) of CLL patients and healthy donors. We found that CD8+ cells from CLL patients presensitized with peptides: R9Y (YLQLDAFEV); R13YL (YLVQSLEDV); R15Y (YLKQTLDEL); R17YL (YLQEQLNKI) as well as R19YL (YLIKHVVKL) specifically recognized T2 cells pulsed with respective peptides with the highest frequency for R9Y (90%). In chromium-51 release assays, R9Y-primed CD8+ T cells from CLL patients were able to effectively lyse R9Y-peptide pulsed T2 cells as well as CLL leukemic cells presenting RHAMM in a HLA-A2 restricted manner; in contrast, K562 cells and T2 cells lacking either RHAMM or HLA-A2 expression were not lysed above background levels. In addition, mAb-based MHC I blockade demonstrated that the observed peptide-specific lysis was MHC I-restricted.In conclusion, we defined a novel RHAMM-derived CD8+ restricted, heteroclitical, cryptic epitope R9Y which might represent an interesting target for immunotherapy of CLL. In future clinical administration the R9Y peptide might circumvent tolerance to dominant epitopes and thereby increase efficacy of peptide vaccination. DisclosuresStilgenbauer:GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

The tumor antigens RHAMM and G250/CAIX are expressed in head and neck squamous cell carcinomas and elicit specific CD8+ T cell responses

Despite advances in surgery, radio- and chemotherapy, therapeutic approaches for patients with head and neck squamous carcinoma (HNSCC) need to be improved. Immunotherapies eliciting tumor specific immune responses might constitute novel treatment options. We therefore investigated the expression and immunogenicity of two tumor-associated antigens (TAA) the receptor for hyaluronic acid mediated motility (RHAMM) and carboanhydrase IX (G250/CAIX) in HNSCC patients. Twenty-two HNSCC samples were examined for the expression of RHAMM and G250 by Western blotting and immunohistochemistry, 14/22 samples were tested for HLA-A2 expression by flow cytometry. For 8/22 samples single tumor-cell suspensions were generated, and mixed lymphocyte peptide cultures (MLPC) were performed to evaluate the frequencies of cytotoxic T cells specifically recognizing RHAMM and G250 using Tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays. RHAMM and G250 were expressed in 73 and 80% of the HNSCC samples at the protein level. A co-expression of both TAAs could be detected in 60% of the patients. In 4/8 HLA-A2+ patients, 0.06-0.13% of CD8+ effector T cells recognized Tetramers for RHAMM or G250 and secreted IFNgamma and granzyme B in ELISPOT assays. RHAMM and G250 are expressed at high frequency and high protein level in HNSCCs and are recognized by cytotoxic CD8+ effector T cells. Therefore both TAAs constitute interesting targets for T cell based immunotherapies for HNSCC.

The candidate immunotherapeutical target, the receptor for hyaluronic acid-mediated motility, is associated with proliferation and shows prognostic value in B-cell chronic lymphocytic leukemia

Differential expression of molecules in chronic lymphocytic leukemia (CLL) may define prognostic markers and suitable targets for immunotherapy. Expression of the tumor-associated antigen (TAA) RHAMM (receptor for hyaluronic acid-mediated motility) as well as RHAMM splicing variants was assessed in series of 72 CLL patients. Quantitative reverse transcriptase PCR showed higher RHAMM expression in high-risk CLL patients, as well as in the advanced stages of the disease. CLL cases with a higher RHAMM expression showed a significantly shorter median treatment-free survival. Among patients with mutated immunoglobulin heavy chain genes, an analysis of RHAMM expression enabled to distinguish subgroup of patients with favorable prognosis. In lymph nodes, RHAMM staining correlated with a higher Ki-67 index and CD40L expression. Functionally, stimulation with CD40L enhanced RHAMM expression in CLL. We further characterized RHAMM-specific CD8(+) T cells in patients with CLL, as the expression of TAAs might influence the clinical outcome by the means of immune reactions. The cytotoxic potential of RHAMM-specific T cells was shown against target cells bearing RHAMM-derived epitope as well as against CLL cells expressing RHAMM. In conclusion, RHAMM expression appears to be of prognostic value, as well as may reflect the proliferative capacity of CLL cells, and might therefore represent interesting target for immunotherapy.

Role of RHAMM within the hierarchy of well-established prognostic factors in colorectal cancer

Objective:To compare the independent prognostic effect of a panel of immunohistochemical protein markers in colorectal cancer (CRC) and determine their ranking among the established prognostic factors T stage, N stage,...

The Receptor for Hyaluronic Acid Mediated Motility (RHAMM): Characterization as an Immunotherapeutical Target in Chronic Lymphocytic Leukemia (CLL) and First Results of RHAMM-Derived Peptide Vaccination Trial.

Background and Aims: The increased expression of receptor of hyaluronic acid mediated motility (RHAMM) was noted in CLL. The defective expression of RHAMM-exon4 splice variant that correlated with chromosomal instability was found to bear negative prognostic value in patients with multiple myeloma. Therefore, here we characterized expression of RHAMM with its splice variants and correlated it with prognosis in CLL. Moreover, based on our previous results we initiated a RHAMM-derived R3 peptide vaccination study for patients with B-CLL in early stages of disease.Methods: Messenger RNA expression of RHAMM/CD168 was assessed by quantitative RT-PCR in blood samples from 65 B-CLL patients, at the time of diagnosis. The splice variant expression was measured using fluorescence capillary electrophoresis. Three hundred mcg RHAMM R3 peptide (ILSLELMKL) emulsified with the incomplete Freund's adjuvant (ISA-51) day 3, as well as GM-CSF days 1–5, was administrated four times subcutaneously at a biweekly interval.Results: The real-time RT-PCR results revealed higher RHAMM/TBP expression rates in patients with unmutated IgVH status compared with mutated CLL cases (0.047 vs. 0.029, p=0.012). Both splice variants RHAMMFL as well as RHAMM-exon4 showed significantly higher expression in unmutated CLL cases. Increased expression of RHAMM/TBP was noted in advanced stages of disease. A tendency towards higher RHAMM/TBP expression ratios was observed in B-CLL cases with del11q. CLL patients with a RHAMM/TBP ratio >0.028 showed a significantly shorter median treatment-free survival (TFS) (16 vs. 38 months, p=0.0019). In a bivariate analysis, patients with VH mutated status and lower amounts of RHAMM presented longer TFS compared to unmutated cases with a high RHAMM expression (110 vs. 8 months, respectively). An enhanced RHAMM expression was observed in bone marrow samples of 5 B-CLL patients compared with the results obtained in blood (RHAMM/TBP 0.102 vs 0.032). Similar tendency to higher RHAMM/TBP ratios was observed after CD40L stimulation (RHAMM/TBP 0.025 vs.0.113). In five patients who completed all four doses of R3 peptide vaccine no toxicity related to vaccine was observed. Reduction of leukemic cells was noted in 3 patients but no clinical responses meeting NCI criteria was observed. Immunological responses were observed in patients who achieved hematological improvement. Conclusion: RHAMM/CD168 is a novel LAA in B-CLL patients, and its expression levels are correlated with the clinical course of the disease. RHAMM expression might indicate the proliferative potential of leukemic cells. First results of peptide vaccination using RHAMM-derived peptide showed limited reduction of tumor burden.

Immunological and Clinical Responses in Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS), Multiple Myeloma (MM) and Chronic Lymphocytic Leukemia (CLL) after RHAMM-R3 Peptide Vaccination.

We initiated a phase I/II R3 peptide vaccination for patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) overexpressing the receptor for hyaluronic acid mediated motility (RHAMM). RHAMM is a leukemia associated antigen (LAA) that is strongly expressed in several hematological malignancies and induces humoral and cellular immune responses. In this study, patients with AML, MDS, MM or CLL were included with RHAMM expression but with a limited tumor load or a minimal residual disease. To date, 25 patients were enrolled. The first 12 patients were vaccinated with 300 mcg and further patients with 1000 mcg R3 peptide emulsified with the incomplete Freund's adjuvant. The vaccine was given four times at a biweekly interval and GM-CSF was added for five days each vaccination. Only mild drug-related adverse events were observed such as erythema and induration of the skin at the site of injection. Immunological analysis were performed using enzyme linked immunospot (ELISpot) assays for Interferon gamma and Granzyme B, tetramer staining and chromium release assays. We detected specific immune responses in 70% of patients. In most patients, we found an increase of CD8+/HLA-A2/RHAMM R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in flow cytometry in accordance with an increase of R3-specific CD8+ T cells in ELISpot assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Moreover, we measured IL-2 and IL-10 levels in sera before and after vaccination. While IL-10 levels remained at a rather low level, we detected an increase of IL-2 in four of ten patients who showed also clinical responses. RHAMM transcripts in bone marrow and peripheral blood were quantified by real-time RT-PCR. Responding patients showed a decrease of RHAMM after vaccination. We detected positive clinical effects in several patients with myeloid disorders showing a reduction of blasts in the bone marrow. One MDS patient did not need any longer erythrocyte transfusions. Two patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematological malignancies.

Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypical brain slice model.

Although it is known that malignant astrocytomas infiltrate diffusely into regions of normal brain, it is frequently difficult to identify unequivocally the solitary, invading astrocytoma cell in histopathological preparations or experimental astrocytoma models. The authors describe an experimental system that facilitates the tracking of astrocytoma cells by using nonneoplastic cerebral tissue as the substrate for invasion. Cerebral tissue was cut into 1-mm-thick slices and cultured in the upper chamber of a Transwell culture dish on top of a polyester membrane (0.4-mm pore size) that was bathed in medium supplied by the lower chamber. Two astrocytoma cell lines, U-87 MG (U87) and U343 MG-A (U343), were selected because of their differing basal cell motilities in monolayer cultures. The astrocytoma cells were stably transfected with vectors that expressed green fluorescent protein (GFP), either alone or as a fusion protein with the receptor for hyaluronic acid-mediated motility (RHAMM) in either sense or antisense orientations. Stably transfected clones that had high levels of GFP expression were selected using the direct visualization provided by fluorescence microscopy and fluorescence-activated cell-sorter analysis. The GFP-expressing astrocytoma cell clones were implanted into the center of the brain slice and the degree of astrocytoma invasion into brain tissue was measured at different time points by using the optical sectioning provided by the confocal laser microscope. The authors observed that GFP-expressing astrocytoma cells could be readily tracked and followed in this model system. Individual astrocytoma cells that exhibited green fluorescence could be readily identified following their migration through the brain slices. The GFP-labeled U87 astrocytoma cells migrated farther into the brain slice than the U343 astrocytoma cells. The RHAMM-transfected GFP-labeled astrocytoma cells also infiltrated farther than the GFP-labeled astrocytoma cells themselves. The expression of antisense RHAMM virtually abrogated the invasion of the brain slices by both astrocytoma cell lines. The authors believe that this organotypical culture system may be of considerable utility in studying the process of astrocytoma invasion, not only because it provides a better representation of the extracellular matrix molecules normally encountered by invading astrocytoma cells, but also because the GFP tag enables tracking of highly migratory and invasive astrocytoma cells under direct vision.

THE HYALURONIC ACID RECEPTORS INDUCED BY STRETCH INJURY OF RAT BLADDER IN VIVO AND INFLUENCES SMOOTH MUSCLE CELL CONTRACTION IN VITRO

Purpose Loss of bladder compliance from hypercontractility and fibrosis may represent an injury response to excessive intravesical pressure. Together, interactions between cell and extracellular matrix components regulate cell response to injury and extracellular matrix remodeling. The receptor for hyaluronic acid mediated motility (RHAMM) is a recently described hyaluronic acid binding protein known to influence multiple types of cell extracellular matrix interaction in development, injury and cancer. We evaluate the role of RHAMM in mediating early events in bladder stretch injury. Materials and Methods An acute stretch injury model was used. The rat bladder was injured by hyrodistension inducing gross hematuria. Tissues were analyzed for temporal and spatial expression of RHAMM in the mucosa and detrusor regions by immunostaining, western and reverse transcriptase polymerase chain reaction analyses. The contractile activity of smooth muscle cell primary cultures was analyzed using a gel contraction assay in the presence of peptide fragments known to block RHAMM function. Results Acute hydrodistension caused immediate and significant injury to the bladder, with fracturing of smooth muscle cell bundles, edema and hemorrhage. RHAMM immunolocalized to the mucosa and detrusor within 2 hours of injury, peaking by 5 to 10 hours. A shift from low molecular weight (55 kD.) to high (120 kD.) receptor isoforms was prominent during the peak expression period noted by immunolocalization. RHAMM messenger ribonucleic acid increased only slightly (40%) by 5 hours after injury. Smooth muscle cell primary cultures actively initiated and maintained the contraction of collagen gels by more than 75% of baseline in vitro. Blocking RHAMM function significantly inhibited the ability to less than 25% of smooth muscle cells to contract the gels in vitro. Conclusions Increased expression of RHAMM is an early event precipitated by stretch injury to the bladder. Since extracellular matrix hyaluronic acid is found early in tissue repair responses, its receptor RHAMM may be mediating initial bladder responses to stretch injury, some of which (contraction) may be experimentally blocked in vitro. Since the receptor directly regulates protein kinase signaling which is turn mediates smooth muscle cell contraction and collagen synthesis, further studies of RHAMM function in bladder pathology are warranted.