Mounting evidence indicates that Smad proteins are required for TGF beta signaling, but the way(s) in which Smad proteins propagate this signal is unclear. We found that two human Smad proteins (Smad3 and Smad4) could specifically recognize an identical 8 bp palindromic sequences (GTCTAGAC). Tandem repeats of this palindrome conferred striking TGF beta responsiveness to a minimal promoter. This responsiveness was abrogated by targeted deletion of the cellular Smad4 gene. These results define a novel biochemical property of Smad proteins that is likely to play a direct role in the biologic responses to TGF beta and related ligands.

DPC4 (MADH4, SMAD4) is a tumor suppressor gene inactivated by allelic loss in approximately 55% of pancreatic adenocarcinomas. Unfortunately, it can be technically very difficult to detect the inactivation of DPC4 at the genetic level because genetic analyses require the microdissection of relatively pure samples of neoplastic and normal tissues. This is especially true for pancreatic adenocarcinomas, which elicit vigorous, non-neoplastic, stromal responses. Immunohistochemical labeling can overcome this hurdle because it preserves morphological information. We therefore studied the expression of the DPC4 gene product in 46 cancers, including 5 cancer cell lines by Western blot analysis and 41 primary periampullary adenocarcinomas by immunohistochemistry. The status of exons 1-11 of the DPC4 gene in all 46 of the cancers had been previously characterized at the molecular level, allowing us to correlate Dpc4 expression directly with gene status. Three cell lines had wild-type DPC4 genes, and Dpc4 expression was detected in all three by Western blot. The two cell lines with homozygously deleted DPC4 genes did not show Dpc4 protein by Western blot analysis. Immunohistochemical labeling revealed that 17 (94%) of the 18 primary adenocarcinomas with wild-type DPC4 genes expressed the DPC4 gene product, whereas 21 (91%) of 23 primary adenocarcinomas with inactivated DPC4 genes did not. Cases in which there was discordance between the immunohistochemical labeling and the genetic analyses were reanalyzed genetically, and we identified a deletion in exon 0 of DPC4 in one of these cases. This is the first report of a mutation in exon 0 of DPC4 in a pancreatic cancer. The contrast between the strong expression of Dpc4 by normal tissues and the loss of expression in the carcinomas was highlighted in several cases in which an infiltrating cancer was identified growing into a benign duct. These observations suggest that immunohistochemical labeling for the DPC4 gene product is an extremely sensitive and specific marker for DPC4 gene alterations in pancreatic carcinomas. The sensitivity and specificity of immunohistochemical labeling for Dpc4 in other periampullary carcinomas has yet to be determined.

Smad4 (DPC4) is a candidate tumor suppressor gene that has been hypothesized to be critical for transmitting signals from transforming growth factor (TGF) ␤ and related ligands. To directly test this hypothesis, the Smad4 gene was deleted through homologous recombination in human colorectal cancer cells. This deletion abrogated signaling from TGF-␤, as well as from the TGF-␤ family member activin. These results provide unequivocal evidence that mutational inactivation of Smad4 causes TGF-␤ unresponsiveness and provide a basis for understanding the physiologic role of this gene in tumorigenesis.

Yeast two-hybrid screening was used to explore novel proteins that interact with a breast tumor or metastasis suppressor, SYK (spleen tyrosine kinase). The screening yielded NHERF (Na + /H + exchanger regulatory factor, also known as NHERF1 or EBP-50) that binds to the interdomain B of SYK. NHERF is an estrogen-responsive gene that encodes an inhibitory factor for epithelial Na + /H + exchanger isoform 3 (NHE3). We found intragenic mutation of the NHERF gene accompanied by loss of heterzygosity (LOH) in B3% (3/85) of breast cancer cell lines and primary breast tumors. Mutations occurred at the conserved PDZ domains at NHERF NH 2 -terminus that bound to SYK, or at its COOH-terminus motif that binds to MERLIN, the product of Neurofibromatosis 2 (NF2) tumor suppressor gene. NHERF tumorigenic mutations decreased or abolished its interaction with SYK or MERLIN, suggesting a pathway link among these three molecules that may play a critical role in mammary neoplastic progression. Primary breast tumors with LOH at the NHERF locus had clinical presentations of higher aggressiveness, indicating that deregulated NHERF signaling may be associated with disease progression. Moreover, the LOH was inversely correlated with SYK promoter methylation, suggesting that NHERF and SYK may transduce a common suppressive signal. Taken together, the results indicated NHERF to be a candidate tumor suppressor gene in human breast carcinoma that may be interconnected to the SYK and MERLIN suppressors.

HAb18G ⁄ CD147, a member of the immunoglobulin family enriched on the surface of tumor cells, is reported to be correlated with invasion, metastasis, growth, and survival of malignant cells. Here, we found that annexin II, a 36-kDa Ca 2+ -and phospholipid-binding protein and in vivo substrate for tyrosine kinase and PKC, is a new interaction protein of HAb18G ⁄ CD147 in human hepatocellular carcinoma (HCC) cells. In the present study, we explored the unclear role of annxin II in HCC invasion and migration and the interaction effects between HAb18G ⁄ CD147 and annexin II. Our data show that downregulation of annexin II in HCC cells significantly decreased the secretion of MMP, migration ability, and invasive potential, and affected the cytoskeleton rearrangement of tumor cells. The MMP-2 level and invasive potential of HCC cells were regulated by both annexin II and HAb18G ⁄ CD147. Also, interaction effects exist between the two molecules in tumor progression, including MMP-2 production, migration, and invasion. These results suggest that annexin II promotes the invasion and migration of HCC cells in vitro, and annexin II and HAb18G ⁄ CD147 interact with each other in the same signal transduction pathway working as a functional complex in tumor progression. (Cancer Sci 2010; 101: 387-395) I nvasion and migration are two central processes of malignant tumors that usually lead to tumor-associated death. Such migratory and invasive events are regulated by different proteolytic enzymes, including serine protease, cysteine protease, and metalloproteases.(1) These proteolytic enzymes degrade the extracellular matrix (ECM) and basement membrane surrounding blood vessels. During metastasis, cancer cells penetrate through the degraded basement membrane and ECM, become implanted in the underlying tissues, and subsequently form secondary tumors.(2) Among the proteases, MMP are mainly regulated by tumor-stroma interactions via CD147, a highly glycosylated cell surface transmembrane protein belonging to the immunoglobulin superfamily.(3) CD147 was first identified as a factor shed from the surface of tumor cells responsible for stimulating MMP-1 production by fibroblasts. One of the important and most studied functions of CD147 is its role in induction of MMP production via cell interactions -thus the derivation of its other name: extracellular matrix metalloproteinase inducer (EMMPRIN).(4) Accumulating evidence suggests a prominent role for CD147 in mediating interactions both between tumor cells themselves and between tumor cells and 'hijacked' host stromal cells to promote a number of events during cancer progression.(5) Although the detailed mechanisms through which CD147 regulates the phenomenon are not yet known, it is clear that CD147 is a major mediator of malignant cell behavior. Our team previously developed the anti-hepatocellular carcinoma (HCC) mAb HAb18 by using a cell suspension extracted from fresh human HCC tissues to immunize BALB ⁄ c mice and prepare hybridomas.(6-8) Its antigen, HAb18G, was identified ...

MKK4, located in close proximity to p53 gene, is thought to be a tumor suppressor and a metastasis suppressor gene. A low-rate MKK4 gene alteration has been found in a few tumor types, including breast and pancreatic. A suppressor activity for prostate and ovarian tumor metastasis has also been suggested. To understand the pathobiologic roles of MKK4 in tumorigenesis, we examined the phenotypic changes in response to perturbation of the MKK4 expression in breast and pancreatic cancer cell lines. Ectopic expression of MKK4 by adenoviral delivery in MKK4-negative cancer lines stimulated the cell proliferation and invasion, whereas knockdown of MKK4 expression by small interference RNA in an MKK4-positive breast cancer cell line, MDA-MB-231, resulted in decreased anchorage-independent growth, suppressed tumor growth in mouse xenograft model, and increased cell susceptibility to apoptosis brought by stress signals such as serum deprivation. These results argue that MKK4 functions as a prooncogenic molecule instead of a suppressor in breast and pancreatic tumors.

The gene product of spleen tyrosine kinase (SYK) has been implicated in the suppression of breast cancer invasion. We previously reported that SYK expression is lost in a subset of breast cancer; primarily by methylation-mediated gene silencing. In our study, we explored the possibility of using a DNA methyltransferase inhibitor, 5-aza-2 -deoxycytidine (AZA), to suppress breast cancer cell invasion by restoring SYK expression. We found that AZA treatment reestablished the expression of SYK(L) that was accompanied by suppression of the invasion capacity of SYK-negative cells. This invasion inhibition was not due to global cellular toxicity since this treatment did not affect overall cell proliferation. This decreased invasiveness by AZA treatment was diminished by piceatannol, a SYK inhibitor, suggesting that SYK play a significant role in AZA-inducible invasion suppression. SYK promoter hypermethylation was found infrequent in pathologically normal mammary tissues or benign lesions (<5%). In contrast, SYK methylation was frequently identified in ductal carcinoma in situ (ϳ45%) and invasive ductal carcinoma (47% in node-negative and 40% in node-positive cases), indicating that the hypermethylation of SYK occurs at a stage prior to the development of invasion phenotypes. All these results suggested a potential use of SYK methylation as a valuable biomarker to detect early cancerous lesions and support the use of AZA as a new reagent to the management of advanced breast cancer. © 2004 Wiley-Liss, Inc. Key words: DNA methylation; breast cancer; spleen tyrosine kinase; invasionThe pathologic features of breast cancer follow a sequential progression through benign proliferative hyperplasia, to hyperplasia with atypia, to carcinoma in situ and eventually to invasive and metastatic disease. 1,2 The clinical manifestation of breast cancer is the progression of disease to stages characterized by local invasion of the auxiliary lymph nodes, followed by metastasis primarily to the lung, bone and liver. Invasion and metastasis are two closely related steps that involve changes in the physical coupling of cells to their microenvironment such as alterations of E-cadherin or ␤-catenin and activation of extracellular proteases. 3,4 The development of local invasion and distant metastasis are strongly associated with low therapeutic efficacy and poor survival. 5,6 The clinical value is thus evident to develop diagnostic biomarkers prior to the establishment of invasive disease and to discover effective treatments.Because of the ineffectiveness of traditional therapeutic measures for breast cancer at its advanced stage, the search for compounds against cancer invasion has long been an intensive research area. Despite continued efforts, no effective measure has been discovered to prevent or treat this type of invasive disease. Targeting some of the most obvious molecules, such as metalloproteinase, has proved ineffective, 7 probably because the metalloproteinase family comprises a large number of highly homologous molecules. Oth...

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.