A pituitary gene encodes a protein that produces differentiation of breast and prostate cancer cells

Abstract

A cDNA clone of 1.1 kb encoding a 108-aa polypeptide was isolated from a human pituitary cDNA library by expression cloning. This protein was named tumor differentiation factor (TDF). The recombinant TDF protein and a 20-aa peptide, P1, selected from the ORF of the gene, induced morphological and biochemical changes consistent with differentiation of human breast and prostate cancer cells. Fibroblast, kidney, hepatoma, and leukemic lymphocytic cell lines were unaffected. Breast and prostate cancer cells aggregated in spheroidlike structures within 24 h of exposure to TDF. This effect was abrogated by a specific affinity-purified rabbit polyclonal anti-P1 Ab. E-cadherin expression was increased in a dose-dependent manner by TDF. Treatment of MCF7 cells with TDF led to production of a lactalbumin-related protein. Peptide P1 significantly decreased the growth of androgen-independent DU145 prostate cancer in severe combined immunodeficient mice. The presence of TDF protein in human sera was detected by the anti-P1 Ab, suggesting a role of TDF in endocrine metabolism. The fact that all activities of TDF can be mimicked by a peptide derived from the encoding TDF sequence opens the possibility of therapeutic applications.M alignant transformation is characterized by the uncoupling of proliferation and differentiation leading to continuing multiplication of cells and the impairment of their ability to progress to complete normal differentiation. Restoration of differentiation of malignant cells has long been considered a potential therapy of cancer (1-5).We have previously reported that an alkaline extract of rat mammosomatotropic tumor MtTW10 induced morphological and biochemical changes in rat and human breast cancer cells indicative of differentiation. Within 24 h of the exposure in vitro, the pituitary tumor extract (PTE) led to aggregation of breast cancer cells, polarization of organelles, the formation of cell junctions and basement membrane, synthesis of mRNA for casein and lactalbumin, and overexpression of E-cadherin (6). The PTE was also effective on prostate cancer cells, inducing cellular changes and overexpression of E-cadherin and prostate-specific antigen.Because this activity was not reproduced by any of the known pituitary hormones or other growth factors, studied alone or in combination, we assumed that the PTE contained a factor, which we called tumor differentiation factor (TDF). Our attempts to isolate and purify this factor by using conventional chromatographic procedures were unsuccessful. Now, using expression cloning, we report the isolation of a human cDNA clone encoding a protein responsible for this tumordifferentiating activity. We have sequenced the clone. Computer analysis indicates that it encodes a polypeptide with no significant homology to any other known sequence in the GenBank database. Our results support the proposition that TDF is a previously unrecognized pituitary factor. Materials and MethodsPituitary cDNA Library. A cDNA library prepared from a growth hormone-producing human ...

Structural investigation of tumor differentiation factor

“…The gene sequence was according to Ref. [2] and was tagged with FLAG at the N-terminus and with His at the C-terminus. The selection antibiotic was ampicillin.…”

“…So far, the existence of TDF was demonstrated only through indirect methods such as identification of the tdf mRNA through in situ hybridization [2] or of its protein product through WB (Fig. 1).…”

“…TDF induces the differentiation of human breast and prostate cancer cells in vitro and in vivo [1], [2]. However, TDF does not have any morphological or biochemical differentiation effect on fibroblasts or on kidney, hepatoma, and leukemic lymphocytic cell lines.…”

“…The molecular mechanism through which TDF induces cell differentiation is not known [1][2][3][4]. To shed some light into TDF's function, our laboratory isolated and characterized potential TDF receptor (TDF-R) candidates from breast cancer cell lines, nonbreast cancer cell lines, and from fibroblasts [3], [4].…”

“…For example, it has been demonstrated that TDF is glycosylated by N-linked oligosaccharides [2], but its amino acid sequence contains no glycosylation site. In addition, nothing is known about the role of TDF in the blood and why it is secreted.…”