Loss of transcription factor KLF5 in the context of p53 ablation drives invasive progression of human squamous cell cancer

Abstract

Squamous cell cancers account for more than half of all human cancers, and esophageal cancer is the sixth leading cause of cancer death worldwide. The majority of esophageal squamous cell carcinomas have identifiable p53 mutations, yet the same p53 mutations are found at comparable frequencies in precancerous dysplasia, indicating that transformation requires additional somatic changes yet to be defined. Here, we show that the zinc finger transcription factor Krüppel-like factor 5 (KLF5) transactivates NOTCH1 in the context of p53 mutation or loss. KLF5 loss limited NOTCH1 activity and was sufficient on its own to transform primary human keratinocytes harboring mutant p53, leading to the formation of invasive tumors. Restoration of NOTCH1 blocked transformation of KLF5-deficient and p53-mutant keratinocytes. Although human dysplastic epithelia accumulated KLF5, KLF5 expression was lost concurrently with NOTCH1 in squamous cell cancers. Taken together, these results define KLF5 loss as a critical event in squamous cell transformation and invasion. Our findings suggest that KLF5 may be a useful diagnostic and therapeutic target in esophageal squamous carcinomas and possibly more generally in other cancers associated with p53 loss of function.

Figure 1

KLF5 knockdown with p53 mutation or loss decreases NOTCH1 expression and signaling. (A) By Western blot, both full-length (FL) and intracellular (IC) NOTCH1 were decreased when KLF5 shRNA was expressed along with p53^R175H or p53 shRNA. (B) NOTCH1 mRNA was also reduced by KLF5 knockdown in the context of p53^R175H mutation or p53 suppression. Non-silencing shRNA was used as a control for p53 shRNA and for KLF5 shRNA. (C) Expression of the Notch target gene HES1 was markedly reduced by KLF5 knockdown in keratinocytes with mutant p53^R175H or p53 shRNA. (D) KLF5 knockdown also decreased activity of a CBF1 luciferase reporter, a marker of Notch pathway activation, in cells containing p53^R175H or p53 shRNA.

Figure 2

KLF5 and p53 bind and coordinately regulate NOTCH1. (A-B) As expected, quantitative chromatin immunoprecipitation (ChIP) revealed binding of p53 (A) to NOTCH1. However, when p53 binding was lost, with p53^R175H or p53 shRNA, there was a reciprocal rise in KLF5 binding (B) to the same region. (C-D) NOTCH1 luciferase reporter (N1PR) was activated by KLF5 (C) and inhibited by KLF5 suppression (D) in EPC2-hTERT-p53^R175H cells. Mutations within the p53 binding sites of N1PR (N1PRmt) failed to abolish these effects, but NOTCH1 responsiveness to KLF5 was abolished by additional mutations in two putative KLF5 binding sites.

Figure 3

KLF5 knockdown with p53^R175H promotes keratinocyte transformation, tumor growth, and invasion. (A). By 14 days, numerous cells with both KLF5 knockdown and p53^R175H became spindle-shaped (arrows), compared to the rounded, epithelioid appearance with KLF5 knockdown alone, consistent with a mesenchymal phenotype and EMT. (B) Quantification revealed a statistically significant difference in the number of spindle shaped cells with both KLF5 knockdown and p53^R175H, compared to KLF5 knockdown or p53^R175H alone. (C) Growth in soft agar was seen with both KLF5 knockdown and p53^R175H but not with either KLF5 knockdown or p53^R175H alone. Restoration of Notch signaling with NICD inhibited colony formation in soft agar of EPC2-hTERT cells with KLF5 knockdown and p53^R175H.

Figure 4

KLF5 loss in the presence of mutant p53 leads to tumor growth and invasion in vivo and in vitro. (A) Primary human esophageal keratinocytes with both KLF5 knockdown and p53^R175H but not either alone formed tumors in SCID/NCr mice. (B) Xenograft tumors from keratinocytes with both KLF5 knockdown and p53^R175H had islands of invasive tumor cells containing abundant eosinophilic cytoplasm and large hyperchromatic and pleomorphic nuclei with numerous mitoses (arrowheads), in a background of desmoplastic stroma. Tumor cells also showed intercellular bridges (arrows), consistent with squamous cell differentiation. Magnification=100×. (C) In organotypic culture, primary human esophageal keratinocytes with p53^R175H underwent normal stratification; cells with KLF5 knockdown failed to stratify normally, consistent with the roles of KLF5 in proliferation and migration of non-transformed keratinocytes. When KLF5 was silenced and p53^R175H was expressed, keratinocytes were less mature with increased nuclear-cytoplasmic ratios and microinvasion, characterized by invasive nests and small, detached islands of neoplastic squamous epithelia (arrows). Expression of NICD in cells with KLF5 suppression and p53^R175H blocked transformation and invasion. Magnification=200×.

Figure 5

KLF5 is lost in human esophageal squamous cell cancer. (A) KLF5 was normally expressed in a gradient with highest levels in basal and suprabasal cells. In high-grade dysplasia, strong nuclear KLF5 expression was seen throughout esophageal epithelia; expression was lost in invasive esophageal squamous cell cancers. Magnification=100×. (B) Quantitatively, the average score for KLF5 staining per cell increased by 70% from normal (NML, n=6) epithelia to high-grade dysplasia (HGD, n=5), but decreased by 45% and 70%, respectively from NML or HGD to invasive squamous cell cancer (CA, n=6). Furthermore, 37% of NML, 11% of HGD, and 59% of CA cells had no staining, while the percentage with scores of 2 or greater was 17% for NML, 39% for HGD, and 6% for CA. (C) Esophageal squamous cell cancer lines lost KLF5, NOTCH1, and p21^Waf1/Cip1 expression by quantitative real-time PCR. (D) KLF5 expression in esophageal squamous cell cancer lines was highly correlated with NOTCH1 and p21^Waf1/Cip1 expression.

Figure 6

A model for KLF5 and p53 in the regulation of NOTCH1 and esophageal squamous cell carcinogenesis.