TGFBI, an extracellular secreted matrix protein, was originally implicated as a regulator of cell adhesion and migration. More recently, down-regulation of TGFBI expression has been reported to be involved in the development of human tumors, including lung, breast, ovarian, prostate, embryonic rhabdomyosarcoma, insulinoma, and mesenchymal tumors [14
]. Loss of TGFBI expression has also been observed in neoplastic transformation in CHO cells and papillomavirus-immortalized human bronchial epithelial cells [3
The physiological role of TGFBI is still largely unknown. It has been reported that the embryonic expression of TGFBI is particularly strong in the mesenchyme of many tissues throughout all stages of development [34
]. In addition, immunohistochemical analysis has demonstrated that TGFBI proteins are deposited in ECM and in cytoplasm and nuclei. Analyses of medium and matrix fractions displayed a protein at 70–74
kDa, and nuclear extracts showed a 65
kDa reactive protein band [35
]. We also found that TGFBI protein localized not only in cell culture medium and cytoplasm, but also in the nuclei of TGFBI-transfected tumor cells and immortalized epithelial cells (Met-5A cells and MCF-10
F cells). The diverse distribution of TGFBI suggests that the functions of TGFBI may not be limited to its role as a component of ECM.
The FAS1 domains of TGFBI have been shown to inhibit tumor angiogenesis and tumor growth and to promote apoptosis. This is also consistent with a tumor suppressor role for TGFBI [36
]. Recent evidence has shown that TGFBI expression causes significantly higher sensitivity to apoptotic induction by upregulation of IGFBP3 [29
]. It also repressed tumor cell invasion, possibly by suppressing the PI3K/Akt/mTOR signaling pathway [37
]. Loss of TGFBI expression is frequent in human cancer and it has been causally related to acquisition of tumorigenic phenotype in asbestos-treated immortalized human bronchial epithelial cells. In this study, by re-introduction of TGFBI into tumor cell lines MDA-MB-231 and NCI-H28, which have naturally low levels of TGFBI, we substantiated the role of TGFBI as a tumor suppressor and more importantly discovered previously unknown portions of its underlying mechanism.
Our data show that TGFBI significantly reduced cell growth rate, plating efficiency, and anchorage-independent growth. These parameters are often used to assess the fundamental characteristics linked to the functions of oncogenes and tumor suppressors. The results are consistent with proposed biological functions of TGFBI and results obtained from this and previous studies [1
]. Cell cycle progression through G1 phase into S phase is a major checkpoint for cells during proliferation. Dysregulation of the G1/S transition may arrest the cells in quiescence or drive them into nonstop proliferation, depending on the specific scenario. A number of oncogenes and tumor suppressors affect the G1/S transition directly or indirectly, notably cyclin A1, p21, and p53 [38
]. Data from this study demonstrates that TGFBI upregulates p53 and p21. This suggests that the inhibitory effect of TGFBI on this checkpoint may be related to these two molecules.
Earlier, our group presented evidence that TGFBI deficiency can lead to mutations, chromosomal fragmentation, and genetic instability, which in turn promotes tumor development. Similarly, ablation of TGFBI increases the frequency of chromosomal aberration and micronuclear formation, as observed in fibroblast cells isolated from TGFBI knock-out mice. However, these cells also showed more proliferation and earlier entry into S-phase entry than those of wild-type mice [39
]. In this study we did not check for genetic instability but rather precisely reproduced the evidence of TGFBI’s inhibitory effects on cell proliferation, transformation, and G1/S transition using a different model, which strongly supported the conclusion that TGFBI is a tumor suppressor. It may execute its function by modulating or interacting with other cell cycle effectors, ultimately leading to unchecked cell proliferation and malignant transformation.
Cellular senescence is defined as a state of irreversible arrest in cell division after a period of serial proliferation in normal diploid cells [40
]. It can also serve as a stress protective response. It can be triggered by a number of sensing mechanisms, such as telomere shortening, epigenetic derepression of the INK4a/ARF locus that encodes two physically linked tumor suppressor proteins p16/p14, and DNA damage [41
]. p16 has been shown to inhibit the ability of cyclin D1 to hinder S-phase entry, which is one of the possible mechanisms involved in the regulation of cellular senescence [42
]. Escaping senescence is a prerequisite for cell immortalization and transformation [43
]. We therefore asked if TGFBI’s inhibitory effect on cellular transformation (anchorage-independent growth and malignancy) is related to its modulation of senescence. To our surprise, an enhanced senescence accompanied by the expression of TGFBI was evidenced by the increased levels of SA-β-gal, a classic marker of cellular senescence.
Elevation of telomerase activity, another sign of senescence, however, exhibited different pattern in mesothelioma and breast cancer cells. In NCI-H28 cells, telomerase activity increased significantly with the expression of TGFBI, which directs cells into senescence. The loss of TGFBI is therefore believed to contribute to the escape of cells from senescence. However, TGFBI did not affect telomerase activity in MDA-MB-231 cells. The expression of p16 and p14 showed no significant difference between TGFBI-expressing and control cells. Homozygous deletion of the p16 gene has been reported in 85% of mesothelioma cell lines, including NCI-H28 cells and 22% of primary tumor specimens [46
]. This makes it difficult to assess the functional association between TGFBI and p16. Other mechanisms may be involved in controlling the process, p21 and p53 are potential candidates [48
]. In both types of cells, p21 and p53 were both up-regulated upon TGFBI expression. Our results clearly showed that SA-β-gal and telomerase activity were both up-regulated by TGFBI re-expression. This may suggest that TGFBI carries out its inhibitory functions on cellular senescence involving p21 and p53.
Further results derived from in vivo
substantiated the role of TGFBI as a tumor suppressor. After implanting cells with TGFBI and leaving others without, we analyzed the onset, incidence, and volume of the resulting tumors in mice, in order to assess the tumor suppressive effect of TGFBI. Although TGFBI did not completely block the formation of tumors derived from injection of MDA-MB-231 cells, the onset of tumor formation was delayed, tumor volume was greatly reduced, and the number of tumors decreased dramatically. This is in accordance with our previous data, which showed that TGFBI suppresses tumorigenic phenotypes in lung and human bronchial epithelial cells induced by radiation and asbestos [14
]. Unfortunately, both TGFBI-expressing and vector control meosthelioma cells failed to produce progressively growing tumors even at 5 months after cell inoculation. We are not sure what the exact reason for this may be. One explanation could be that the residual immunity of nude mice may still be able to reject some types of cells. One alternative means of evaluating these phenomena would be to use SCID mice, which lack both T and B lymphocytes, unlike nude mice, which only lack T cells. For further evaluation of the inhibitory role of TGFBI on a molecular level, tissue slides dissected from tumors in each group were stained with the nuclear antigen ki67, which serves as a marker of cellular proliferation capacity [23
]. The number of ki67-positive cells inversely correlated with the level of TGFBI expression; the more ki67-positive cells observed in the vector control groups, the stronger the evidence that TGFBI diminishes the ability of cells to proliferate and therefore inhibits tumorigenicity in vivo
. We here present strong evidence that unequivocally supports that TGFBI exhibits an inhibitory effect on tumor growth both in vitro
and in vivo
, especially in mesothelioma and breast cancer cells.
Contrary evidence from other groups was brought into our attention. For example, it has been suggested that TGFBI increases the metastatic ability of colon and an ovarian cancer cell lines [51
] In addition, TGFBI has been shown to be over-expressed in pancreatic cancer, renal cell carcinoma and glioblastoma [53
]. It is likely that TGFBI protein may function in multiple ways depending on tissue type and tumor microenvironment. TGFBI
gene is a downstream target of transforming growth factor beta (TGF-β) that inhibits the proliferating of normal epithelial cells and functions as a tumor suppressor in early tumorigenesis as well as a tumor promoter in later stage of tumor progression. This stage-specific dual functional role of TGFBI in cancer represents an emerging paradigm whereas the mechanism behind is not well understood. We are planning to expand our research to more type of cell lines and clinical samples. Our particular focus will be on the questions left unanswered by this and other reports.