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 8 genes selected for validation, 7 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, overexpression 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.

The epithelial-mesenchymal transition (EMT) and its reversal, MET, are fundamental processes involved in tumor cell invasion and metastasis. SEMA3F is a secreted semaphorin and tumor suppressor downregulated by TGFβ1 and ZEB1-induced EMT. Here we report that NRP2, the high-affinity receptor for SEMA3F and a co-receptor for certain growth factors, is upregulated during TGFβ1-driven EMT in lung cancer cells. Mechanistically, NRP2 upregulation was TβRI-dependent and SMAD-independent, occurring mainly at a post-transcriptional level involving increased association of mRNA with polyribosomes. ERK and AKT inhibition blocked NRP2 upregulation, while RNAi-mediated attenuation of ZEB1 reduced steady-state NRP2 levels. Additionally, NRP2 attenuation inhibited TGFβ1-driven morphologic transformation, migration/invasion, ERK activation, growth suppression and changes in gene expression. In a mouse xenograft model of lung cancer, NRP2 attenuation also inhibited locally invasive features of the tumor and reversed TGFβ1-mediated growth inhibition. In support of these results, in human lung cancer specimens with the highest NRP2 expression were predominantly E-cadherin negative. Furthermore, the presence of NRP2 staining strengthened the association of E-cadherin loss with high-grade tumors. Together, our results demonstrate that NRP2 contributes significantly to TGFβ1-induced EMT in lung cancer.