Positive Feedback Regulation between KLF5 and XPO1 Promotes Cell Cycle Progression of Basal like Breast Cancer

Abstract

Basal-like breast cancer (BLBC), overlapping with the subgroup of estrogen receptor (ER), progesterone receptor (PR), and HER2 triple-negative breast cancer, has the worst prognosis and limited therapeutics. The XPO1 gene encodes nuclear export protein 1, a promising anticancer target which mediates nucleus-cytoplasm transport of nuclear export signal containing proteins such as tumor suppressor RB1 and some RNAs. Despite drugs targeting XPO1 are used in clinical, the regulation of XPO1 expression and functional mechanism is poorly understood, especially in BLBC. This study finds that KLF5 is a transcription factor of XPO1, which increases RB1 nuclear export and cell proliferation in BLBC cells. Furthermore, XPO1 interacts with the RNA-binding protein PTBP1 to export FOXO1 mRNA to cytoplasm and thus activates the FOXO1-KLF5 axis as a feedback. This work demonstrates that XPO1 inhibitor KPT-330 in combination with CDK4/6 inhibitor additively suppressed BLBC tumor growth in vivo. These results reveal a novel positive feedback regulation loop between KLF5 and XPO1 and provide a novel treatment strategy for BLBC.

Keywords: KLF5; RB1; XPO1; basal like breast cancer; cell cycle.

Figure 1

XPO1 promotes BLBC cell proliferation. A) The mRNA expression levels of XPO1 in BLBC clinical samples from GEPIA online database. *p < 0.05.B) The mRNA expression levels of XPO1 in different types of breast cancer from TCGA database. ***p < 0.001.C) The bc‐GenExMiner v4.5 online database was used to analyze XPO1 mRNA expression levels in different breast cancer subtypes. ***p < 0.001. D) The XPO1 protein overexpression in BLBC cells was detected by WB. E) The activity of cells was detected by CCK‐8 assay after XPO1 overexpression(n = 3). ****p < 0.0001. F) XPO1 overexpression in BLBC cells accelerated the cell cycle progression, as detected by flow cytometry (n = 3). G) Quotative results of panel F. *p < 0.05, n. s, not significant. H) XPO1 stable knocked down by shRNA in BLBC cells was detected by WB. I) XPO1 knockdown in BLBC reduced cell viability, as detected by CCK‐8 assays (n = 3). **p <0.01, ***p < 0.001. J) XPO1 knockdown in BLBC cells blocked the cell cycle progression, as detected by flow cytometry (n = 3). K) Quotative results of panel J. *p < 0.05.

Figure 2

KLF5 promotes XPO1 gene transcription and cell proliferation through XPO1 in BLBC. A) The positive association between KLF5 and XPO1 mRNA expression levels in clinical BLBC samples from the TCGA database. B) The expression of KLF5 and XPO1 proteins in BLBC patient tissue samples, as assessed by immunohistochemical (IHC) staining. The images are shown at 5× and 40× magnification (n = 87). C) RNA‐seq data suggest that XPO1 mRNA was positively regulated by KLF5 in HCC1806 cells. D) KLF5 knockdown decreased the XPO1 protein levels in BLBC cells. The protein expression levels of XPO1 were detected by WB. E) KLF5 knockdown decreased the XPO1 mRNA levels in BLBC cells. The mRNA expression levels of XPO1 were detected by RT‐qPCR (n = 3). *p < 0.05, **p < 0.01. F) Dual luciferase reporter plasmids of the full‐length and truncated XPO1 gene promoter were constructed, respectively, and named as, pXPO1, Site1, Site2, Site3. G) The binding of KLF5 to site3 at the XPO1 gene promoter was detected by dual luciferase reporter assays (n = 3). *p < 0.05, n. s, not significant. H) The site3 mutation abolished the activation of the XPO1 gene promoter by KLF5, as detected by dual luciferase reporter assays (n = 3). *p < 0.05, n. s, not significant. I) The binding sites of KLF5 and XPO1 promoter region were detected by ChIP‐qPCR (n = 3). *p < 0.05, n. s, not significant. J) KLF5 knockdown and XPO1 overexpression in BLBC cells were detected by WB. K) XPO1 overexpression can partially rescue KLF5 knockdown induced cell growth inhibition. CCK‐8 assay was used to detect the cell viability (n = 3). **p < 0.01, ****p < 0.0001. L) WB was used to verify the effect of KLF5 knockdown and XPO1 overexpression in HCC1806 cells. M) XPO1 overexpression can partially rescue KLF5 knockdown induced tumor growth inhibition, as measured by tumor volume changes (n = 10). ***p < 0.001. N) XPO1 overexpression can partially rescue KLF5 knockdown induced tumor growth inhibition. Tumor masses were obtained from nude mice.

Figure 3

XPO1 promotes BLBC cell proliferation by transporting RB1 protein out of the nucleus. A) RB1 knockdown in BLBC cells was detected by WB. B) RB1 knockdown promoted BLBC cell proliferation, as measured by the CCK‐8 assays (n = 3). ***p < 0.001, ****p < 0.0001. C) Endogenous XPO1 protein interacts with RB1protein, as measured by Co‐IP assays in BLBC cells. D) XPO1 knockdown did not affect the expression levels of total RB1 protein in BLBC cells, as detected by WB. E) XPO1 knockdown increased the nuclear localization of RB1. Nuclear and cytoplasmic fractionations were collected for WB. F) Knockdown of XPO1 and RB1 in BLBC cells were detected by WB. G) Knockdown of RB1 rescued the cell growth inhibition induced by XPO1 knockdown in BLBC cells, as assessed by CCK‐8 cell growth assays (n = 3). ****p < 0.0001. H) Knockdown of RB1 rescued the cell cycle inhibition induced by XPO1 knockdown in BLBC cells. Cell cycle changes were detected by flow cytometry (n = 3). I) Quotative results of panel H. *p < 0.05, n. s, not significant. J) XPO1 inhibitor (KPT‐330) decreased the XPO1 protein expression levels in BLBC cells, as detected by WB. K) KPT‐330 inhibited BLBC cell growth, as assessed by CCK‐8 cell growth assays (n = 3). *p < 0.05, ****p < 0.0001. L) KPT‐330 did not affect the expression levels of total RB1 protein in BLBC cells, as detected by WB. M) KPT‐330 increased the nuclear accumulation of RB1. N) KPT‐330 significantly inhibited the growth of tumor cells. The tumor volume of nude mice was measured once every 3 days (n = 7). *p < 0.05. O) Treatment of BLBC with KPT‐330 for 18 days resulted in a significant reduction in tumor mass (n = 14). P) Quotative results of panel O. ***p < 0.001.

Figure 4

KLF5 increases Cyclin D1 and XPO1 expression to promote RB1 phosphorylation and nuclear export. A) The protein expression levels of XPO1, RB1 and KLF5 in normal breast epithelium and breast cancer cell lines, as measured by WB. B) TNFα induced both KLF5 and XPO1 protein expression in BLBC cells, as detected by WB. C) KLF5 is essential for TNFα to induce XPO1 protein expression in BLBC cells, as detected by WB. D) KLF5 knockdown decreased the protein expression levels of XPO1, Cyclin D1, FGF‐BP1 and p‐RB1 in BLBC cell lines, as detected by WB. E) KLF5 knockdown increased the nuclear localization of RB1, as detected by WB. F) KLF5 knockdown and XPO1 overexpression in BLBC cells were verified by WB. G) XPO1 overexpression rescued KLF5 knockdown induced the nuclear localization of RB1, as detected by WB. H) TNFα increased the protein expression of Cyclin D1, pRB1, XPO1 and FGF‐BP1 in BLBC cells, as detected by WB. I) After TNFα stimulation of BLBC cells, nuclear export of RB1 was promoted, Nuclear and cytoplasmic fractionations were collected for WB.

Figure 5

XPO1 promotes KLF5 expression by binding to PTBP1 and FOXO1 mRNA nuclear export in BLBC cells. A) XPO1 knockdown decreased the protein expression levels of FOXO1 and KLF5, but did not affect the protein expression levels of YB1 and PTBP1 in BLBC cells, as detected by WB. B) XPO1 knockdown decreased the mRNA expression levels of KLF5, but did not affect the mRNA expression levels of FOXO1 in BLBC cells, as detected by RT‐qPCR (n = 3). *p < 0.05, n. s, not significant. C) FOXO1 overexpression rescued XPO1 knockdown induced KLF5 expression decrease in BLBC cells, as detected by WB. D) XPO1 knockdown in BLBC cells increased the nuclear localization of FOXO1 mRNA, as detected by RT‐qPCR (n = 3). *p < 0.05. E) XPO1 knockdown in BLBC cells increased the nuclear localization of PTBP1 protein, as detected by WB. F) KPT‐330 decreased the total protein expression levels of FOXO1 and KLF5 in BLBC cells, as detected by WB. G) KPT‐330 increased the nuclear localization of PTBP1 and RB1 proteins, as detected by WB. H) KPT‐330 increased the nuclear localization of FOXO1 mRNA, as determined by RT‐qPCR (n = 3). **p < 0.01. I) PTBP1 knockdown decreased the protein expression levels of FOXO1, KLF5 and XPO1, as detected by WB. J) PTBP1 knockdown decreased the mRNA expression levels of KLF5, but not FOXO1, as detected by RT‐qPCR (n = 3). *p < 0.05, n. s, not significant. K) PTBP1 knockdown increased the nuclear localization of FOXO1 mRNA, as determined by RT‐qPCR (n = 3). *p < 0.05. L) The binding between the endogenous XPO1 and PTBP1 proteins was examined by Co‐IP assays in BLBC cells. M) XPO1 bound to the FOXO1 mRNA and PTBP1 protein in BLBC cells. IP was performed with the anti‐XPO1 Ab. Proteins were examined by WB and mRNA was detected by RT‐qPCR (n = 3). *p < 0.05, **p < 0.01. N) PTBP1 bound to the FOXO1 mRNA and XPO1 protein in BLBC cells. IP was performed with the anti‐PTBP1 Ab. Proteins were examined by WB and mRNA was detected by RT‐qPCR (n = 3). *p < 0.05, **p < 0.01.

Figure 6

CDK4/6 inhibitor Palbociclib, in combination with XPO1 inhibitor KPT‐330, has an additive therapeutic effect on BLBC. A) The expression levels of XPO1, p‐RB1 and RB1 proteins in BLBC cells treated with CDK4/6 inhibitor (Palbociclib) and KPT‐330 were detected by WB. B) The combination of Palbociclib and KPT‐330 additively inhibited BLBC cell proliferation, as measured by CCK‐8 assays (n = 3). **p < 0.01, ****p < 0.0001. C) The combination of Palbociclib and KPT‐330 additively induced cell cycle G1 phase arrest. The cell cycle changes were detected by flow cytometry. D) Quotative results of panel C (n = 3). *p < 0.05, n. s, not significant. E, F) BLBC cells were treated with various concentrations of Palbociclib and KPT‐330 for 72h. The inhibition percentages of cell viabilities were measured by CCK‐8 assay (n = 3), and combination effects were analyzed using the Online Synergy Finder web application 3.0. G) The combination of Palbociclib and KPT‐330 can significantly inhibit the growth of BLBC tumors. BLBC nude mice were treated by combined administration for 21 days, and tumor volume was measured once every 3 days (n = 5). *p < 0.05, **p < 0.01, ****p < 0.001, n. s, not significant. H) Palbociclib combined with KPT‐330 can significantly reduce the tumor weight of BLBC in nude mice. After 21 days of combined treatment, the nude mice were sacrificed, and the tumor weight was taken (n = 10). I) Quotative results of panel F. *p < 0.05, ****p < 0.0001. J) Diagram of the regulatory mechanism between KLF5 and XPO1 in BLBC.