Abstract
Trefoil factor 3 (TFF3) expression is positively associated with advanced clinicopathological features of mammary carcinoma (MC). Herein, we provide evidence for a functional role of TFF3 in oncogenic transformation of immortalized, but otherwise normal human mammary epithelial cells (HMECs), namely, HMEC-hTERT, MCF10A, and MCF12A. Forced expression of TFF3 in immortalized-HMECs enhanced cell proliferation, cell survival, anchorage-independent growth, produced highly disorganised three-dimensional (3D) acinar structures and generated tumours in immunocompromised mice. Forced expression of TFF3 in immortalized-HMECs stimulated STAT3 activity that was required for TFF3-stimulated cell proliferation, survival, and anchorage-independent growth. TFF3 specifically utilised STAT3 activity to govern a transcriptional program, which was required for TFF3-stimulated oncogenic transformation of immortalized-HMECs, including transcriptional upregulation of CCND1 and BCL2. siRNA-mediated depletion or functional inhibition of STAT3 significantly inhibited the TFF3-stimulated transcription of CCND1 and BCL2 and oncogenicity in immortalized-HMECs. Furthermore, DOX-inducible expression of TFF3 in HMEC-hTERT cells also permitted anchorage-independent growth and produced disorganized acinar structures in 3D Matrigel culture. Removal of DOX-induced expression of TFF3 in HMEC-hTERT cells, previously grown with DOX, resulted in efficient normalisation of the disorganized acinar architecture and attenuated cell viability in Matrigel culture. Cumulatively, these findings suggest that TFF3 is a potent oncogene and its increased expression along with hTERT in HMECs is sufficient to produce oncogenic transformation.
Highlights
A combination of cell proliferation and cell survival have been postulated to provide a platform for the oncogenic transformation of normal cells[1,2]
Soluble whole cellular extracts were run on an SDS-PAGE and immunoblotted as described in materials and methods. β-actin was used as an input control for cell lysate
(see figure on previous page) Fig. 6 CCND1 and BCL2, both are essential for trefoil factor 3 (TFF3)-driven oncogenic transformation of immortalized-human mammary epithelial cells (HMECs). a Western blot analysis was used to assess the levels of CCND1 and BCL2 in immortalized-HMECs with forced expression of TFF3 and their vector control cells after siRNAmediated depletion of CCND1 or BCL2
Summary
A combination of cell proliferation and cell survival have been postulated to provide a platform for the oncogenic transformation of normal cells[1,2]. HMEC-hTERT, MCF10A and MCF12A cells were transfected with the pIRESneo3-TFF3 expression construct to generate the corresponding stable cell lines with forced expression of TFF3; a pIRESneo[3] construct was used as vector control as described in Materials and methods[7,8]. The levels of TFF3 expression in immortalized-HMECs with forced expression of TFF3 and their vector control cells was demonstrated using a RT-PCR and b western blot. C Representative phase-contrast microscopic images of immortalized-HMECs with forced expression of TFF3 and their vector control cells on monolayer culture. D Representative confocal laser scanning microscopy images of immortalized-HMECs with forced expression of TFF3 and their vector control cells cultured on Matrigel (2D). Forced expression of TFF3 in immortalized-HMECs significantly abrogated apoptotic cell death as a consequence of serum deprivation when compared to their vector control cells. The role of TFF3 in the oncogenic transformation process is not defined
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