ZEB1-responsive genes in non-small cell lung cancer

Abstract

The epithelial to mesenchymal transition (EMT) is a developmental process enabling epithelial cells to gain a migratory mesenchymal phenotype. In cancer, this process contributes to metastases; however the regulatory signals and mechanistic details are not fully elucidated. Here, we sought to identify the subset of genes regulated in lung cancer by ZEB1, an E-box transcriptional repressor known to induce EMT. Using an Affymetrix-based expression database of 38 non-small cell lung cancer (NSCLC) cell lines, we identified 324 genes that correlated negatively with ZEB1 and 142 that were positively correlated. A mesenchymal gene pattern (low E-cadherin, high Vimentin or N-cadherin) was significantly associated with ZEB1 and ZEB2, but not with Snail, Slug, Twist1 or Twist2. Among eight genes selected for validation, seven were confirmed to correlate with ZEB1 by quantitative real-time RT-PCR in a series of 22 NSCLC cell lines, either negatively (CDS1, EpCAM, ESRP1, ESRP2, ST14) or positively (FGFR1, Vimentin). In addition, over-expression or knockdown of ZEB1 led to corresponding changes in gene expression, demonstrating that these genes are also regulated by ZEB1, either directly or indirectly. Of note, the combined knockdown of ZEB1 and ZEB2 led to apparent synergistic responses in gene expression. Furthermore, these responses were not restricted to artificial settings, since most genes were similarly regulated during a physiologic induction of EMT by TGF-β plus EGF. Finally, the absence of ST14 (matriptase) was linked to ZEB1 positivity in lung cancer tissue microarrays, implying that the regulation observed in vitro applies to the human disease. In summary, this study identifies a new set of ZEB-regulated genes in human lung cancer cells and supports the hypothesis that ZEB1 and ZEB2 are key regulators of the EMT process in this disease.

Figure 1. RNA screening in NSCLC cell lines

RNA expression was measured by quantitative real-time RT-PCR in a series of 22 NSCLC cell lines, two NHBE cultures and two immortalized human airway primary cell lines (BEAS2B and FC6625-2 3KT). Cells were ranked from left to right according to ZEB1 mRNA expression. Values are expressed as percent of the geometric mean between GAPDH and actin. The experiment was done twice in duplicate. Bars = SD. Numbers 1–4 indicate H358, A549, H460 and H157 cell lines, respectively, discussed later in the text.

Figure 2. Correlations of protein levels with ZEB1 in NSCLC

For each cell line, 10 μg total protein were loaded on a 4–15% polyacrylamide gel. Cell lines were ranked on the gel according to ZEB1 mRNA from low to high levels (left to right). Western blots were performed with indicated antibodies. Molecular weights are provided in kDa. β-actin was used as a loading control. Among these cell lines, those with known mutations of EGFR and sensitivity to an EGFR inhibitor include H3255, HCC4006, H1650 and HCC2279 [48]. In addition, those with IC50s of 1 μM or less in a proliferation assay include H358 and H322 [48].

Figure 3. E-cadherin, ST14 and EpCAM proteins are decreased by exogenous ZEB1 or ZEB2 in H358 cells

Cells stably transfected with inducible myc-tagged ZEB1 or ZEB2, or with vector (ctl) were grown for 5 days and concurrently induced with doxycycline (100 ng/ml) for 0, 1, 3 or 5 days. Protein lysates were analyzed by Western blot for induction of ZEB1 (A) or ZEB2 (B) using anti-myc antibodies. E-cadherin, ST14 and EpCAM were detected by corresponding antibodies. The asterisk indicates a variable background band. β-actin provided a loading control.

Figure 4. E-cadherin, CDS1 and EpCAM are increased by ZEB1 or ZEB2 knockdown in H157 cells

siRNAs targeting ZEB1, ZEB2 or a non-targeting control were transfected into H157 cells. Cells were harvested for analysis 5 days post-transfection. Protein lysates were examined by Western blot using the indicated antibodies. β-actin provided a loading control.

Figure 5. ZEB1-responsive genes are similarly regulated by TGF-β plus EGF treatment

(A) A549 and H358 cells were treated during 48 h with 10 ng/ml TGF-β plus 50 ng/ml EGF. Actin was stained by phalloidin. (B) Expression of ZEB1, ZEB2, Snail and (C) ZEB1 regulated genes was measured by quantitative real-time RT-PCR. Values are expressed as percent of GAPDH.

Figure 6. ST14 positively correlates with E-cadherin but negatively with ZEB1 in lung tumors

(A) Examples of two tumors with either high (upper panel) or low (lower panel) ZEB1 level and corresponding immunostaining for E-cadherin and ST14. Dashed line: separation between tumor and stroma; T: Tumor compartment; S: Stromal compartment; scale bar = 100 μm. (B) Distribution of tumors for E-cadherin or ST14 staining. For ZEB1 nuclear staining, tumors with a score > 5 were considered positive (as only a low number of cells are positive for ZEB1). For E-cadherin and ST14, tumors with scores > 10 were considered positive. (C) Distribution of tumors stained for ZEB1 and compared with E-cadherin or ST14. (B, C) Results are presented as number of tumors.