Overexpression of the RHAMM gene by transfection into fibroblasts is transforming and causes spontaneous metastases in the lung. H-ras-transformed fibrosarcomas transfected with a dominant suppressor mutant of RHAMM exhibit a so-called revertant phenotype and are completely nontumorigenic and nonmetastatic. Conversely, fibroblasts stably expressing low levels of RHAMM as a result of antisense transfection are resistant to ras transformation. Collectively, these results indicate that RHAMM acts downstream of ras. The loss of functional RHAMM ablates signaling within focal adhesions, in particular changes in focal adhesion kinase phosphorylation, and as a result these focal adhesions are unable to turn over in response to hyaluronan. These results provide evidence of the oncogenic potential of a novel extracellular matrix receptor and establish a functional link between transformation by ras and signaling within focal adhesions that are required for transformation by this oncogene.

An expression vector was constructed in which TGF‐beta 1 was placed under the control of the metallothionein promoter. Cys223 and Cys225 in the TGF‐beta 1 propeptide were converted to serines, mutations which result in dissociation of the pro‐peptide and secretion of bioactive TGF‐beta 1 [Brunner, A.M., Marquardt, H., Malacko, A.R., Lioubin, M.N. and Purchio, A.F. (1989) J. Biol. Chem., 264, 13660–13664]. A fibrosarcoma was transfected with this plasmid and a clone (17.18) was selected in which TGF‐beta 1 mRNA was able to be induced six‐fold following zinc sulphate treatment. These cells increased the secretion of bioactive TGF‐beta 1 14‐fold and exhibited a coincidental increase in jun‐B mRNA expression, suggesting that secreted TGF‐beta 1 was acting to induce this early response gene by autocrine activation. Following zinc sulphate induction, the tumor cells became progressively more motile and able to invade collagen gels. In contrast to parental tumor not bearing the TGF‐beta 1 expression vector, zinc sulphate stimulation of clone 17.18 enhanced collagenase IV and procathepsin L mRNA levels and enhanced the secretion of many collagenolytic proteases into the medium. Since the action of TGF‐beta generally decreases proteolysis by suppression of protease transcription, we compared the response of normal parental fibroblasts to ras‐transformed fibrosarcomas and confirmed that TGF‐beta could greatly enhance collagenase IV and procathepsin L mRNA levels while having little effect on non‐transformed fibroblasts. These experiments indicate that induction of TGF‐beta secretion can enhance motility and protease production through autocrine activation, thus increasing the invasion potential of fibrosarcomas.

Abstract. TGF-/3 is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-/3-stimulated locomotion and show that TGF-B triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-Bt and by stimulating the production of bioactive TGF-/3~ in tumor cells transfected with TGF-B~ under the control of the metallothionein promoter. TGF-/31-induced locomotion was suppressed by antibodies that prevented HA/RI-IAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-B,-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-~/with panspecific TGF-/3 antibodies and by inhibition of TGF-/3~ secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-/3, and the pathway by which TGF-~/stimulates locomotion uses the HA receptor RHAMM and HA.

Since the small heat shock protein hsp27 enhances both growth and drug resistance in breast cancer cells, and is a bad prognostic factor in certain subsets of breast cancer patients, we have characterized the transcriptional regulation of hsp27, with the long-term goal of targeting its expression clinically. The majority of the promoter activity resides in the most proximal 200 bp. This region contains an imperfect estrogen response element (ERE) that is separated by a 13-bp spacer that contains a TATA box. Gel-shift analysis revealed the binding of a protein (termed HET for Hsp27-ERE-TATA-binding protein) to this region that was neither the estrogen receptor nor TATA-binding protein. We cloned a complete cDNA (2.9 kb) for HET from an MCF-7 cDNA library. To confirm the identity of the HET clone, we expressed a partial HET clone as a glutathione S-transferase fusion protein, and showed binding to the hsp27 promoter fragment in gel-retardation assays. The HET clone is almost identical to a recently published scaffold attachment factor (SAF-B) cloned from a HeLa cell cDNA library. Scaffold attachment factors are a subset of nuclear matrix proteins (NMP) that interact with matrix attachment regions. Analyzing how HET could act as a regulator of hsp27 transcription and as a SAF/NMP, we studied its subnuclear localization and its effect on hsp27 transcription in human breast cancer cells. We were able to show that HET is localized in the nuclear matrix in various breast cancer cell lines. Furthermore, in transient transfection assays using hsp27 promoter-luciferase reporter constructs, HET overexpression resulted in a dose-dependent decrease of hsp27 promoter activity in several cell lines.

Since the small heat shock protein hsp27 enhances both growth and drug resistance in breast cancer cells, and is a bad prognostic factor in certain subsets of breast cancer patients, we have characterized the transcriptional regulation of hsp27, with the long-term goal of targeting its expression clinically. The majority of the promoter activity resides in the most proximal 200 bp. This region contains an imperfect estrogen response element (ERE) that is separated by a 13-bp spacer that contains a TATA box. Gel-shift analysis revealed the binding of a protein (termed HET for Hsp27-ERE-TATA-binding protein) to this region that was neither the estrogen receptor nor TATA-binding protein. We cloned a complete cDNA (2.9 kb) for HET from an MCF-7 cDNA library. To confirm the identity of the HET clone, we expressed a partial HET clone as a glutathione S-transferase fusion protein, and showed binding to the hsp27 promoter fragment in gel-retardation assays. The HET clone is almost identical to a recently published scaffold attachment factor (SAF-B) cloned from a HeLa cell cDNA library. Scaffold attachment factors are a subset of nuclear matrix proteins (NMP) that interact with matrix attachment regions. Analyzing how HET could act as a regulator of hsp27 transcription and as a SAF/NMP, we studied its subnuclear localization and its effect on hsp27 transcription in human breast cancer cells. We were able to show that HET is localized in the nuclear matrix in various breast cancer cell lines. Furthermore, in transient transfection assays using hsp27 promoter-luciferase reporter constructs, HET overexpression resulted in a dose-dependent decrease of hsp27 promoter activity in several cell lines.