C Rhamm and Tumor Progression

As we have noted earlier, the tissue remodeling that occurs with wound repair resembles that occurring during neoplastic progression (56-59). In particular, the presence of chronic tissue fibrosis, associated with hyper-expression of both CD44 and RHAMM appears to provide favorable conditions for sustaining neoplastic conversion and progression. The similarity between the two processes is reflected by the fact that RHAMM mRNA expression is specifically upregulated in tumors and the appearance of short RHAMM isoforms, particularly those that appear on the cell surface also correlates with tumor progression.

Serological identification of antigens by recombinant expression cloning, SEREX, identified antibodies against RHAMM in acute myeloid leukemia (42%), chronic myeloid leukemia (31%), melanoma (83%), renal cell carcinoma (40%), breast cancer (67%) as well as ovarian carcinoma (50%) (132), and realtime PCR revealed a 1-13.6 fold increase of RHAMM expression in 97% of colon cancer (133). Furthermore, because of the presence of mononucleotide repeat sequences in the coding region, RHAMM is frequently mutated in subsets of colorectal cancers with defects in mismatch repair genes, suggesting a selective pressure for the accumulation of RHAMM mutations (134). Intrigu-ingly, several reports suggest that small tumor subsets are responsible for hyperexpression of both CD44 and RHAMM in breast cancers (90,135). Further, subsets of tumor cells from breast primary tumors that hyper-express CD44 also express stem/progenitor markers and are several hundred times more tumorigenic upon transplantation into immune-compromised mice than tumor cells that do not highly express these markers (62). Similarly, immuno-staining of breast cancer samples identified subsets of cells with high RHAMM expression (90,135) (Fig. 6) and their presence in a primary tumor was significantly associated with poor clinical outcome and with an occurrence of lymphatic metastasis (90). Possible hyper-expression of either CD44 or RHAMM confers a selective advantage to tumor cells that permits colonization, and part of this ability may be related to suppression of pro-apoptotic pathways. For example, RHAMM hyperexpression strongly correlates with erk kinase hyper-expression, a MAP kinase that has been shown to act on the HA-regulated signaling pathway that confers resistance to anchorage dependent apoptosis or anoikis (136-139). Elevated RHAMM has been reported for other tumor types as well. For example in endometrial carcinoma, 100% of tumors in patients with lymph node involvement were positive for RHAMM expression whereas RHAMM expression was found in 50.7% of tumors in patients without lymph node involvement and 13% of normal control tissue (140). As for wound repair, smaller, possibly activated isoforms of RHAMM are predominantly found in tumor tissue. RT-PCR analysis identified full length RHAMM in 49% of random c-DNA clones isolated from multiple myeloma patients (88). A smaller RHAMM isoform missing exon 4, RHAMM"4 was found in 47% of c-DNA clones

whereas only one out of eight normal donors expressed RHAMM (88).

Figure 6 RHAMM localization in breast tumor sections. Paraffin sections of breast tumor samples were stained with anti-RHAMM antibodies. Staining intensity varied between tumors isolated from different patients, but the presence of foci of RHAMM over-expressing tumor cells is prognostic of poor outcome. Foci shown in (A) (arrows) contain nuclear (arrow) and cytoplasmic (arrowhead) staining. Image of another tumor shown in (B) has little RHAMM staining and no foci of RHAMM-hyperexpressing tumor cells for comparison with A. Magnification: (A) 580 X , (B) 360 X .

Furthermore, a study by Greiner et al. (132) demonstrated RHAMM expression in 100% of acute myeloid leukemia, 83% of chronic myeloid leukemia and 100% of renal cell carcinoma patients and expression of RHAMM"48 was found in all RHAMM-positive samples. Comparison of RHAMM expression between astrocytoma cell lines and tissues with normal astrocytes and brain tissue revealed a 70 kDa isoform in addition to the 86 kDa full length RHAMM in cancerous cells or tissues (141). Furthermore, a splice variant missing exon 4 was predominantly expressed in colon tumors compared to normal tissue (142). These results link RHAMM mRNA and protein hyper-expression to human neoplasia and suggest that small protein forms of RHAMM, previously shown to be transforming in murine cells in vitro (89), predominate during neoplastic progression, thus probably representing activated forms of RHAMM.

The clinical correlation between RHAMM expression and tumors suggests that this gene plays key functions in either neoplastic conversion and/or progression. The possibility that RHAMM might play a key role in both processes was first suggested by the demonstration that overexpression of short RHAMM forms transformed 10T1/2 and 3T3 fibroblasts to tumors that were metastatic in tail vein assays. Conversely, expression of either a dominant inhibitory form of RHAMM that cannot bind to HA or soluble recombinant RHAMM forms that compete with cell surface RHAMM (CD168) for HA-binding, blocked rasmediated transformation (89). The relevance of these in vitro studies to tumor formation originating in vitro has recently been confirmed by studies using mice in which the RHAMM gene has been deleted by homologous recombination. RHAMM — /— mice were crossed with mice heterozygous for a mutation in the Adenomatous Polyposis Coli (APC) tumor suppressor gene that blocks the tumor suppressor function of this scaffold protein and which results in elevated levels of beta-catenin protein and, as a consequence, development of aggressive fibromatosis (desmoid) tumors. The APC mutation in this transgenic mouse line differs from that of the 'min' mouse, which is predisposed to aggressive intestinal tumors. Although upper intestinal tract pre-neoplastic polyps are formed in the APC transgenic line, these do not proceed to frank neoplasms over the life span of the mice. Instead, the mice die prematurely from desmoid tumors (143). Loss of RHAMM significantly reduced both the number of desmoid tumors and the size of the tumors, the latter providing a measure of the invasiveness of the desmoid tumor (86). This defect was associated with reduced proliferation of RHAMM — /— tumor cells in response to serum supplements and to PDGF, when the cells were sub-confluent. At high culture confluence a difference in proliferation was not observed. Interestingly, the absence of RHAMM had no effect on the number of pre-neoplastic polyps of the upper intestinal tract (86). While gastrointestinal tumors are derived from epithelial cells, desmoid tumors consist of mesenchymal fibroblastic cells resembling cells that predominate in the granulation phase of wound healing, a stage in wound repair where HA plays a key role (26,144). Collectively, these results indicate a role for RHAMM in the neoplastic transformation of mesenchymal cells and for a predominance of RHAMM function in sparse culture conditions such as those most likely found during wound repair and during tumor invasion and metastasis. These results also raise the intriguing possibility that RHAMM function may predominate following EMT of parenchymal cells, a process that is associated with aggressive tumors and poor clinical outcome.

Several studies have addressed how RHAMM might contribute to cell proliferation. For example, Mohapatra et al. (55) showed that soluble recombinant RHAMM protein, down-regulation of RHAMM function by expression of a dominant negative mutant or antisense, results in G2M arrest of H-ras transformed fibroblasts and suppression of tumor formation as a result of decreased expression of Cdc2/CyclinB1. Maxwell et al. (82) showed that overexpression of full length RHAMM or RHAMM containing a deletion of a putative Cdc2 phosphorylation site in HeLa or Jurkat cells leads to cell cycle arrest and accumulation of cells in prometaphase/metaphase or prophase and abnormal mitotic spindle formation. Although it cannot be excluded that the observed chromosome spindle breakdown is the result of an artificially high RHAMM expression, these results suggest that either increased or decreased RHAMM expression/function blocks cell proliferation. Furthermore, full-length RHAMM may perform a dual inhibitory/stimulatory role in proliferation, unlike the shorter RHAMM forms, which seem to predominantly stimulate. This would be consistent with constitutive expression of shorter RHAMM forms in cancer. The study of Maxwell et al. (82) suggested that RHAMM plays a role in genomic stability although this possibility would be better analyzed by expressing activated RHAMM forms at lower levels than were used in this study. Hyper-expression of several proteins, e.g., beta-catenin, has been shown to promote genomic instability via artifactual processes, i.e., high protein levels result in abnormal accumulation in atypical sub-cellular compartments (145,146). In any event, the accumulation of additional mutations or changes in the cell's stromal microenvironment might allow tumor cells to increasingly rely upon RHAMM as an ECM receptor. This would result in a progressively aggressive tumor, similar to the aggressiveness of CD44 — /— spleenocytes that rely upon RHAMM for functions associated with inflammation and which, in the absence of CD44, result in a strongly enhanced destruction of normal tissues.

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