Inhibition of Hypoxia-Induced Cell Motility by p16 in MDA-MB-231 Breast Cancer Cells

Abstract

Our previous studies indicated that p16 suppresses breast cancer angiogenesis and metastasis, and downregulates VEGF gene expression by neutralizing the transactivation of the VEGF transcriptional factor HIF-1α. Hypoxia stimulates tumor malignant progression and induces HIF-1α. Because p16 neutralizes effect of HIF-1α and attenuates tumor metastatic progression, we intended to investigate whether p16 directly affects one or more aspects of the malignant process such as adhesion and migration of breast cancer cells. To approach this aim, MDA-MB-231 and other breast cancer cells stably transfected with Tet-on inducible p16 were used to study the p16 effect on growth, adhesion and migration of the cancer cells. We found that p16 inhibits breast cancer cell proliferation and migration, but has no apparent effect on cell adhesion. Importantly, p16 inhibits hypoxia-induced cell migration in breast cancer in parallel with its inhibition of HIF-1α transactivation activity. This study suggests that p16's ability to suppress tumor metastasis may be partially resulted from p16's inhibition on cell migration, in addition to its known functions on inhibition of cell proliferation, angiogenesis and induction of apoptosis.

Keywords: HIF-1a; breast cancer; hypoxia; migration/motility; p16.

Figure 1

Induction of transgene by doxycycline (Dox) in breast cancer cells stably transduced with a retroviral-mediated Tet-on system. 4T1 (A and B) and JygMC(A) (C and D) cells stably infected with Lenti-Tet-on GFP were treated with 1 μg/ml doxycycline (Dox, a tetracycline analogue) for 72 h (A-D). The cells were then observed under a fluorescent microscope for transgene GFP expression. The same image of the cells (4T1, A and B; JygMC(A), C and D, respectively) at both fluorescent (A and C) and light-contrast (B and D) images are shown. E and F. Immunohistochemcal (IHC) staining for p16 protein on MDA-MB-231 cells stably infected with Lenti-Tet-on p16 (MDA/Tet-on p16). MDA/Tet-on p16 cells were treated either without (E) or with (F) 1 μg/ml Dox for 72 h. The cells (E and F) were then IHC stained by primary anti-p16 antibody. The dark brown color indicates p16 protein (F).

Figure 2

p16 inhibits breast cancer cell proliferation. The breast cancer cells MDA/Tet-on p16 were incubated with or without 1 μg/ml Dox for 5 days and the cell numbers were counted. The results represent the data from at least two independent experiments, each performed in triplicate.

Figure 3

p16 does not appear to affect breast cancer cell adhesion. The breast cancer cells MDA/Tet-on p16 (or MDA/p16) and MDA/Tet-on GFP (or MDA/GFP) (A) or 4T1/Tet-on p16 (or 4T1/p16) and 4T1/Tet-on GFP (or 4T1/GFP) (B) were incubated with or without 1 μg/ml Dox for 72 h. The cells were then harvested and used for adhesion assay as described in M & M section. The results represent the data from at least two independent experiments, each performed in triplicate. The differences between with and without Dox treatment groups, as well as between Tet-on p16 and Tet-on GFP groups are not significant (p>0.05).

Figure 4

p16 inhibits HIF-1α transcriptional activity under both hypoxia and normoxia conditions. MDA/Tet-on p16 cells were treated with or without 1 µg/ml Dox and cotransfected with pVEGF/Luc and phRLuc-TK. The Dual-Luciferase Assay Kit was used to analyze the luciferase activity of cell extracts. The normalized luciferase activity was represented as ratio of Firefly luciferase activity (pVEGF/luc) over Renilla luciferase activity (phRLuc-TK). The results represent the data from three independent experiments, each performed in duplicate. Some error bars are too small to be seen in this scale.

Figure 5

Hypoxia induced VEGF promoter transactivation and its inhibition by YC-1 involve HRE, the HIF-1α binding region of the promoter. MDA-MB-231 cells were transfected with pMAP11WT and pMAP11mut, followed by either normoxic or hypoxic conditions, and luciferase activity analysis as described in M & M section. A serial concentration of YC-1, a HIF-1α inhibitor, was added in the medium of cell samples under hypoxia in order to inhibit the hypoxia-induced HIF-1α transactivation activity. The normalized data is presented. The results represent the data from at least two independent experiments, each performed in duplicate.

Figure 6

Hypoxia increased cell migration/motility in breast cancer cells. The breast cancer cells 4T1/Tet-on GFP cells were incubated with or without 1 μg/ml Dox for 72 h, followed by 24 h in hypoxic or normoxia incubation prior to be used for cell migration assay as described in M & M section. The results represent the data from at least two independent experiments, each performed in triplicate.

Figure 7

p16 inhibits hypoxia-induced cell migration. (A) The breast cancer cells MDA/Tet-on p16 cells were incubated with or without 1 μg/ml Dox for 72 h, followed by cell migration assay as described in M & M section in normoxic and hypoxic conditions. (B) 4T1/Tet-on p16 (or 4T1/p16) cells were incubated with or without 1 μg/ml Dox for 72 h, followed by cell migration assay in hypoxic conditions. The results represent the data from at least two independent experiments, each performed in triplicate.