Suppression of KIF3A inhibits triple negative breast cancer growth and metastasis by repressing Rb-E2F signaling and epithelial-mesenchymal transition

Abstract

Triple negative breast cancer (TNBC) displays higher heterogeneity, stronger invasiveness, higher risk of metastasis and poorer prognosis compared with major breast cancer subtypes. KIF3A, a member of the kinesin family of motor proteins, serves as a microtubule-directed motor subunit and has been found to regulate early development, ciliogenesis and tumorigenesis. To explore the expression, regulation and mechanism of KIF3A in TNBC, 3 TNBC cell lines, 98 cases of primary TNBC and paired adjacent tissues were examined. Immunohistochemistry, real-time PCR, western blot, flow cytometry, short hairpin RNA (shRNA) interference, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation techniques, transwell assays, scratch tests, and xenograft mice models were used. We found that KIF3A was overexpressed in TNBC and such high KIF3A expression was also associated with tumor recurrence and lymph node metastasis. Silencing of KIF3A suppressed TNBC cell proliferation by repressing the Rb-E2F signaling pathway and inhibited migration and invasion by repressing epithelial-mesenchymal transition. The tumor size was smaller and the number of lung metastatic nodules was lower in KIF3A depletion MDA-MB-231 cell xenograft mice than in the negative control group. In addition, KIF3A overexpression correlated with chemoresistance. These results suggested that high expression of KIF3A in TNBC was associated with the tumor progression and metastasis.

Keywords: KIF3A; Rb-E2F signaling; Triple negative breast cancer; epithelial-mesenchymal transition; metastasis.

Figure 1

KIF3A mRNA and protein expressions were higher in triple negative breast cancer (TNBC) than in adjacent tissues. A, B, The KIF3A mRNA level was detected by real‐time RT‐PCR. C, D, The KIF3A protein level was detected by western blot assays. GAPDH and β‐actin were used as control, **P < 0.01

Figure 2

KIF3A was more highly expressed in triple negative breast cancer (TNBC) than in adjacent tissues by immunohistochemistry assay. A‐D, KIF3A was more highly expressed in cancer cells (A, B) than adjacent tissues (C, D). E, F, Moreover, the cancer cells metastasizing to lymph nodes (E) showed stronger KIF3A expression than the corresponding primary cancer cells (F). DAB (brown) served as chromogen (A, C and E 100×; B, D and F 200×)

Figure 3

The silence and overexpression efficiency of KIF3A was detected. A, The cell lines MDA‐MB‐231, BT20, MDA‐MB‐468 and BT549 were detected by western blot for the expression of KIF3A, with both MDA‐MB‐231 and BT549 showing strong expression level. B, MDA‐MB‐231 and BT549 cells were chosen to deplete KIF3A using KIF3A‐shRNA (1#, 2# and 3#).The KIF3A‐shRNA 3# and 2# were used in the next experiment because of the higher efficiency in silencing of KIF3A. C, MDA‐MB‐231 and BT549 cells were detected to deplete KIF3A using KIF3A‐shRNA (3#) by real‐time RT‐PCR. D, MDA‐MB‐468 cells were used to overexpress KIF3A by KIF3A expression plasmid, and western blot analysis showed that the expression peak of KIF3A appeared at 48 h after KIF3A‐pEX transfection

Figure 4

Silencing of KIF3A inhibits tumor cell growth and G1/S transition by repressing Rb‐E2F signaling. A, Cell proliferation was analyzed by MTT assay. Absorbance was measured at 490 nm. The data were presented as the means of six separated experiments, each performed in triplicate. B, Colony formation results of KIF3A depletion and overexpression were photographed and cell colony numbers are illustrated in a histogram. C, Flow cytometry results show the cell phase distribution of TNBC cell lines. Three independent experiments were conducted. *P < 0.05, **P < 0.01 D, The expressions of Phospho‐Rb, E2F1, Cyclin E1, Cyclin D1 and P21 were detected by western blot in MDA‐MB‐231, BT549 and MDA‐MB‐468 cells, respectively

Figure 5

Silencing of KIF3A suppresses tumor growth on triple negative breast cancer (TNBC) cell xenograft. A, Images of Scr‐shRNA and KIF3A‐shRNA (3#) MDA‐MB‐231 cell xenograft tumor. B, C, Tumor growth curve and average weight were recorded and counted. *P < 0.05, **P < 0.01 D, Immunohistochemistry (IHC) staining in the tumor from KIF3A‐shRNA (3#) (400×) and control group (400×) is shown

Figure 6

Silencing of KIF3A inhibits cell migration and invasion via epithelial‐to‐mesenchymal transition (EMT). A, B, Transwell and invasion assay showed the migrated and invasive cells of MDA‐MB‐231 and BT549 in Scr‐shRNA and KIF3A‐shRNA(3#). C, Images of scratch wound healing and comparison of migration distance of MDA‐MB‐231 and BT549 cells among Scr‐shRNA and KIF3A‐shRNA (3#) were detected by scratch test. D, E, The migration and invasive abilities of Vector and KIF3A‐pEX group cells were detected by migration and invasion assay in MDA‐MB‐468. Data were mean ± SD values from three experiments, each performed in triplicate. **P < 0.01. F, The morphology of Scr‐shRNA and KIF3A‐shRNA (3#) group cells was shown and the morphology of Vector and KIF3A‐pEX group cells was shown. G, The expressions of ZEB1, E‐cadherin, vimentin, MMP‐2, MMP‐9 and KIF3A were detected by western blotting

Figure 7

KIF3A knockdown suppresses triple negative breast cancer (TNBC) metastasis in vivo. Six‐week‐old female nude BALB/c mice were injected with 2 × 10⁶ Scr‐shRNA or KIF3A‐shRNA (3#) MDA‐MB‐231 cells via the tail vein. A, B, The body weight and the lung weight of the mice were measured 6 weeks later. **P < 0.01 C, D, Images of lung metastasis and H&E staining (upper: 200×; lower: 400×) are shown. E, The numbers of metastatic nodules in the lung were calculated in 10 randomly selected high‐power fields under a microscope. **P < 0.01 F, Immunohistochemical staining in xenograft (400×)

Figure 8

KIF3A induces doxorubicin‐resistance but not to cisplatin summary. A, C, E, IC50 of doxorubicin was determined using the MTT assay. Cells (2 × 10³) were seeded and exposed to doxorubicin for 48 h. B, D, F, IC50 of cisplatin was determined using the MTT assay. Cells (2 × 10³) were seeded and exposed to cisplatin for 48 h

Figure 9

Schematic representation of KIF3A inducing triple negative breast cancer (TNBC) cell proliferation, migration and metastasis. KIF3A inhibits p21 expression to promote activity of cyclin‐dependent kinases (CDKs). Consequent Rb phosphorylation by Cyclin/CDK complexes leads to the release of Rb‐E2F complexes, resulting in increasing the expression of E2F and cell cycle‐related proteins, which increases cell cycle progression and finally promotes TNBC cell proliferation. KIF3A also leads to increased expression of MMPs and ZEB1, promoting processes of EMT, which finally accelerates TNBC cell migration and metastasis