Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1.

Sonic hedgehog, Patched and Gli are components of a mammalian signalling pathway that has been conserved during evolution and which has a central role in the control of pattern formation and cellular proliferation during development. Here we identify the human Suppressor-of-Fused (SUFUH) complementary DNA and show that the gene product interacts physically with the transcriptional effector GLI-1, can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signalling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signalling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses.

Counter-selection for over-expressed human CD44s in primary tumors versus lung metastases in a mouse fibrosarcoma model.

Human CD44 standard isoform cDNA (hCD44s) was transfected into sis-transformed Balb/c 3T3 cells and into ras-revertant IIIA4 cells (both tumorigenic but nonmetastatic). Transfectants were injected subcutaneously into athymic nude mice to elucidate the functional role of hCD44s over-expression in progression and metastasis. The transfectants (but not parental cells) were capable of lung micrometastasis and of binding exogenously-added hyaluronan. hCD44s protein expression was conserved in lung micrometastases suggesting that it may have been necessary for their formation. In contrast, no hCD44s protein was detected in large subcutaneous (s.c.) tumors but normal levels of murine CD44 were detected. A second round of tumor development, using these two tumor cell classes, demonstrated that hCD44s-nonexpressing s.c. tumor cells re-expressed it in lung micrometastases. Conversely, hCD44s-expressing lung micrometastatic cells, when injected into a second group of mice, downregulated hCD44s expression in order to grow sizable s.c. tumors. S.c. tumor cells still contained the hCD44s gene but its expression was inhibited by epigenetic mechanisms, one of which was shown to be methylation of the hCD44s gene. These studies demonstrate (a) opposing selective pressures on CD44s over-expression for s.c. tumor growth and for metastatic spread to the lung and (b) further credence for the significance of CD44 for metastatic spread of fibrosarcomas. Therefore, CD44s may be a critical component of the metastatic phenotype induced by specific oncogenes.

Oncogene-dependent expression of CD44 in Balb/c 3T3 derivatives: correlation with metastatic competence.

Oncogene-dependent regulation and tumor relatedness of CD44 expression were investigated in Balb/c 3T3 cells and their derivatives transformed with different ras oncogenes (metastatic tumor model) or the human c-sis oncogene (non-metastatic model). Ras transformants using either the Harvey or Kirsten oncogenes expressed high levels of cell surface CD44 protein that bound fluoresceinated hyaluronan (HA). Much lower levels of CD44 were expressed in parental 3T3 cells, ras- revertants generated from Kirsten-transformed cells, or c-sis transformants, confirming the significance of the ras oncogene in this upregulation. To determine whether endogenous HA regulates these parameters, hyaluronidase treatment of ras transformants exposed more cell surface CD44 to anti-CD44 antibody and increased fluoresceinated HA binding; this did not occur with 3T3 or c-sis transformants. CD44 expression and its HA-binding function were conserved in a panel of in vivo primary and lung metastatic tumor cell lines derived from ras transformants. Ras transformants also retained the ability to downregulate CD44 protein levels in confluent cultures which occurred through a translational or post-translational mechanism (as CD44 mRNA levels were not reduced). These results taken together demonstrate that ras-dependent regulation of CD44 may correlate with tumor progression and metastasis in vivo, possibly (although not exclusively) supporting CD44's importance in metastatic progression.

Over-expression of human CD44s in murine 3T3 cells: selection against during primary tumorigenesis and selection for during micrometastasis.

Human CD44 standard isoform (hCD44s) cDNA regulated by a high-expressing promoter was transfected into Balb/c 3T3 cells and the tumorigenic and metastatic capacities of the transfectants were investigated in nude mice at the subcutaneous site. One of three transfectants was tumorigenic. hCD44s expression was lost in the cells of large primary tumors using this tumorigenic clone. These tumors were extremely aggressive giving overt metastases and micrometastases to several sites including mesentery, stomach, liver, diaphragm, pancreas and lung. Micrometastatic cells re-expressed hCD44s, consistent with its importance for early steps in the metastatic cascade. hCD44s was not expressed in overt metastases; most probably the expression was lost during the outgrowth of micrometastases into overt metastatic tumors. Thus hCD44s expression in murine 3T3 cells does induce tumorigenicity in select cases, is not compatible with aggressive outgrowth of primary or secondary tumors, and is advantageous for early steps in metastatic spread. These results suggest that CD44s is an example of a novel type of 'metastasis' molecule that is disadvantageous for tumor growth and is only transiently advantageous during metastatic spreading of tumor cells to distant organs.

Plasticity of CD44s expression during progression and metastasis of fibrosarcoma in an animal model system.

CD44s (standard isoform), which binds hyaluronan (HA), has been analyzed for its significance during fibrosarcoma progression and metastasis in an athymic nude mouse model using Balb/c 3T3 cells transformed with ras or sis oncogenes. While ras (but not sis) transformation leads to upregulated expression of mouse CD44s and HA binding, transfection/overexpression of human CD44s gene (hCD44s) elicited remarkable plasticity of expression during progression and metastasis. In 3T3, hCD44s induced tumorigenesis, HA binding, and micrometastatic competence to lungs and other organs. In tumorigenic (but nonmetastatic) sis transformants or ras-deleted revertants, it also induced micrometastatic competence. Conversely, large primary tumors and overt metastases lost hCD44s expression and HA binding via hypermethylation of hCD44s gene. Tail vein injections revealed that hCD44s greatly increased the efficiency of colonization of the lung microvasculature at the earliest stages. These studies indicate that hCD44s overexpression and possibly its HA binding are critical for conveying metastatic competence but are antagonistic or selected against during aggressive primary tumor or overt metastasis outgrowth. This remarkable plasticity of expression and its consequences offer an ideal system for dissecting the molecular mechanisms operating during fibrosarcoma progression and metastasis.

Upregulation of CD44s in c-sis-transformed balb/c 3T3 cells by autocrine growth factor mechanisms, including PDGF.

Regulation of CD44s by the c-sis oncogene product was investigated. Both CD44s protein and mRNA were comparably upregulated, consistent with some degree of transcriptional regulation; its ligand binding was also activated in confluent but not in sparse cultures of sis-transformed Balb/c 3T3 cells. CD44s was also elevated in confluent cultures of parental 3T3 cells treated with conditioned media from confluent c-sis transformants (but not from 3T3 cells); these media (but not media from ras transformants or parental 3T3 cells) contained platelet-derived growth factor (PDGF)-immunoreactive material. CD44s upregulation by these media could be partially blocked by anti-PDGF antibodies. These media also induced activation via tyrosine autophosphorylation and activation-dependent downregulation of PDGF beta-receptors. sis-transformed 3T3 cells contained low levels of PDGF receptor, but CD44 levels could still be increased in these cells by addition of PDGF. These results suggest that CD44s is upregulated in confluent cultures of c-sis-transformed cells by autocrine growth factors, including PDGF, secreted into the cell's microenvironment.

CD44 protein levels and its biological activity are regulated in Balb/c 3T3 fibroblasts by serum factors and by transformation with the ras but not with the sis oncogene.

CD44s (standard isoform) levels and hyaluronan-binding activity were investigated in Balb/c 3T3 cells and their derivatives transformed with ras or sis oncogenes as a function of serum concentration in the medium. 3T3 cells contained low levels of CD44 and did not bind hyaluronan when grown in medium containing 0.5 or 10% serum. In 5% serum, however, the cells had much higher levels of CD44 and were able to bind hyaluronan. CD44 levels also increased in 3T3 cells restimulated with either 5 or 10% serum after prior maintenance in low serum. In cells restimulated with 5% serum, high levels of CD44 were sustained for at least 72 hr. In cells restimulated with 10% serum, however, the increase in CD44 levels reverted by 48 hr. Transformation of 3T3 cells with ras (but not with sis) oncogene rendered CD44 levels insensitive to serum modulation: ras-transformed cells contained high levels of CD44 and bound hyaluronan at all serum concentrations and at all time points tested. Sis-transformed cells behaved like 3T3 cells in these modulatory changes. Platelet-derived growth factor (PDGF), when supplementing 0.5% serum, mimicked the effects of serum on the levels and hyaluronan-binding capacity of CD44 in 3T3 cells and the CD44-upregulating activity of serum was neutralized by incubation with anti-PDGF antibodies. These data demonstrate that serum factors, specifically PDGF, mediate regulation of CD44 levels in BAlb/c 3T3 cells and that transformation of 3T3 cells by ras renders CD44 expression insensitive to the modulating effects of serum in vitro. These results correlate with the metastatic capacity of these cells in vivo.

Overexpressed human CD44s promotes lung colonization during micrometastasis of murine fibrosarcoma cells: facilitated retention in the lung vasculature.

Normally nonmetastatic murine sis-transformed BALB/c 3T3 cells, transfected with human CD44s gene (hCD44s), acquire spontaneous metastatic capacity to the lung. The mechanism(s) of this facilitated micrometastasis was analyzed in an experimental metastasis model. Human CD44s overexpression promoted the earliest stages severalfold (initial implantation and subsequent stabilization of tumor cells) but was irrelevant for later stages (subsequent outgrowth) of lung experimental micrometastasis. By injecting mixed populations of parental (nonmetastatic) and CD44s-transfected cells, it was shown that cell-cell adhesion between tumor and parental cells was not promoted by hCD44s but that promotion of cell-cell adhesion to lung endothelium or specifically between transfected cells (via hyaluronan) are likely mechanisms. Results obtained with hCD44s-negative primary tumor cells and hCD44s-positive or -negative variants of lung micrometastatic cells (after s.c. injection of transfectants) confirmed the importance of CD44s overexpression for early but not late stages of experimental lung metastasis. Therefore, CD44s represents a metastasis-facilitating molecule that is irrelevant for primary tumor outgrowth but that promotes micrometastasis to the lungs at the very earliest stages.

GLI3 mutations in human disorders mimic Drosophila cubitus interruptus protein functions and localization.

Truncation mutations of the GLI3 zinc finger transcription factor can cause Greig cephalopolysyndactyly syndrome (GCPS), Pallister-Hall syndrome (PHS), and postaxial polydactyly type A (PAP-A). GLI3 is homologous to Drosophila Cubitus interruptus (Ci), which regulates the patched (ptc), gooseberry (gsb), and decapentaplegic (dpp) genes. Ci is sequestered in the cytoplasm and is subject to posttranslational processing whereby the full-length transcriptional activator form (Ci155) can be cleaved to a repressor form (Ci75). Under hedgehog signaling, the Ci155 form translocates to the nucleus whereas in the absence of hedgehog, the Ci75 form translocates to the nucleus. Based on the correlation of GLI3 truncation mutations and the human phenotypes, we hypothesized that GLI3 shows transcriptional activation or repression activity and subcellular localization similar to Ci. Here we show that full-length GLI3 localizes to the cytoplasm and activates PTCH1 expression, which is similar to full-length Ci155. PHS mutant protein (GLI3-PHS) localizes to the nucleus and represses GLI3-activated PTCH1 expression, which is similar to Ci75. The GCPS mutant protein has no effect on GLI3-activated PTCH1 transcription, consistent with the role of haploinsufficiency in this disorder. The PAP-A mutant protein (GLI3-PAP-A) showed less specific subcellular localization but still inhibited GLI3-activated PTCH1 transcription, suggesting it may be a weaker allele than the GLI3-PHS mutation. These data show that GLI3 mutations in humans mimic functional effects of the Drosophila ci gene and correlate with the distinct effects of these mutations on human development.