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.

Loss of SEMA3F occurs frequently in lung cancer and correlates with advanced stage of disease. We previously reported that SEMA3F blocked tumor formation by H157 lung cancer cells in a rat orthotopic model. This was associated with loss of activated A V B 3 integrin, impaired cell adhesion to extracellular matrix components, and down-regulation of phospho-extracellular signal-regulated kinase 1/2 (ERK1/2). These results suggested that SEMA3F might interfere with integrin outside-in signaling. In the present report, we found that SEMA3F decreased adhesion to vitronectin, whereas integrin-linked kinase (ILK) kinase activity was down-regulated in SEMA3F-expressing H157 cells. Exposure to SEMA3F-conditioned medium led to diminution of phospho-ERK1/2 in four of eight lung cancer cell lines, and ILK silencing by small interfering RNA led to similar loss of phospho-ERK1/2 in H157 cells. Moreover, SEMA3F expression (with constitutive and inducible systems) also reduced AKT and signal transducer and activator of transcription 3 (STAT3) phosphorylation independently of ILK-ERK1/2. These signaling changes extended downstream to hypoxia-inducible factor-1A (HIF-1A) protein and vascular endothelial growth factor (VEGF) mRNA levels, which were both reduced in three of four SEMA3F-transfected cell lines. Mechanistically, the effects on HIF-1A were consistent with inhibition of its AKT-driven protein translation initiation, with no effect on HIF-1A mRNA level or protein degradation. Furthermore, when H157 cells were injected s.c. in nude mice, tumors derived from SEMA3F-expressing cells showed lower microvessel density and tumor growth. These results show that SEMA3F negatively affects ILK-ERK1/2 and AKT-STAT3 signaling, along with inhibition of HIF-1A and VEGF. These changes would be anticipated to contribute significantly to the observed antitumor activity of SEMA3F. [Cancer Res 2007;67(18):8708-15]

Although endothelial cell apoptosis participates in the tumor shrinkage after single high-dose radiotherapy, little is known regarding the vascular response after conventionally fractionated radiation therapy. Therefore, we evaluated hypoxia, perfusion and vascular microenvironment changes in an orthotopic prostate cancer model of conventionally fractionated radiation therapy at clinically relevant doses (2 Gy fractions, 5 fractions/week). First, conventionally fractionated radiation therapy decreased tumor cell proliferation and increased cell death with kinetics comparable to human prostate cancer radiotherapy. Secondly, the injection of Hoechst 33342 or fluorescent-dextrans showed an increased tumor perfusion within 14 days in irradiated tumors, which was correlated with a clear reduction of hypoxia. Improved perfusion and decreased hypoxia were not explained by increased blood vessel density, size or network morphology. However, a tumor vascular maturation defined by perivascular desmin+/SMA+ cells coverage was clearly observed along with an increase in endothelial, zonula occludens (ZO)-1 positive, intercellular junctions. Our results show that, in addition to tumor cell killing, vascular maturation plays an uncovered role in tumor reoxygenation during fractionated radiation therapy.

Loss of the 3p21.3-encoded semaphorins, SEMA3B and SEMA3F, is implicated in lung cancer development. Although both antagonize VEGF binding/response to neuropilin (NRP) receptors, in lung cancer lines, SEMA3F is predominantly expressed and preferentially utilizes NRP2. In lung cancer patients, SEMA3F loss correlates with advanced disease and increased VEGF binding to tumor cells. In cell lines, VEGF enhances adhesion and migration in an integrin-dependent manner, and exogenous SEMA3F causes cells to round and lose extracellular contacts. Using retroviral infections, we established stable SEMA3F transfectants in two NSCLC cell lines, NCI-H157 and NCI-H460. When orthotopically injected into nude rats, both control lines caused lethal tumors in all recipients. In contrast, all animals receiving H157-SEMA3F cells, survived to 100 days, whereas all H157 controls succumbed. In H460 cells, which express NRP1 but not NRP2, SEMA3F did not prolong survival. This antitumor effect in H157 cells was associated with loss of activated alpha(v)beta(3) integrin and adhesion to extracellular matrix components. In addition, H157-SEMA3F cells, and parental H157 cells exposed to SEMA3F-conditioned medium, showed loss of p42/p44 MAPK phosphorylation. Thus, in this in vivo lung cancer model, SEMA3F has potent antitumor effects, which may impinge on activated integrin and MAPK signaling.

Gliomas are characterised by local infiltration, migration of tumour cells across long distances and sustained angiogenesis; therefore, proteins involved in these processes are most likely important. Such candidates are semaphorins involved in axon guidance and cell migration. In addition, semaphorins regulate tumour progression and angiogenesis. For cell signalling, class-4 semaphorins bind directly to plexins, whereas class-3 semaphorins require additional neuropilin (NRP) receptors that also bind VEGF 165 . The anti-angiogenic activity of class-3 semaphorins can be explained by competition with VEGF 165 for NRP binding. In this study, we analysed the expressions of seven semaphorins of class-3, SEMA4D, VEGF and the NRP1 and NRP2 receptors in 38 adult glial tumours. In these tumours, SEMA3B, SEMA3G and NRP2 expressions were related to prolonged survival. In addition, SEMA3D expression was reduced in high-grade as compared with low-grade gliomas. In contrast, VEGF correlated with higher grade and poor survival. Thus, our data suggest a function for a subset of class-3 semaphorins as inhibitors of tumour progression, and the prognostic value of the VEGF/SEMA3 balance in adult gliomas. Moreover, in multivariate analysis, SEMA3G was found to be the only significant prognostic marker.

SEMA3F is a secreted semaphorin with potent antitumor activity, which is frequently downregulated in lung cancer. In cancer cell lines, SEMA3F overexpression decreases hypoxia-induced factor 1alpha protein and vascular endothelial growth factor mRNA, and inhibits multiple signaling components. Therefore, understanding how SEMA3F expression is inhibited in cancer cells is important. We previously defined the promoter organization of SEMA3F and found that chromatin remodeling by a histone deacetylase inhibitor was sufficient to activate SEMA3F expression. In lung cancer, we have also shown that ZEB-1, an E-box transcription repressor, is predominantly responsible for loss of E-Cadherin associated with a poor prognosis and resistance to epidermal growth factor receptor inhibitors. In the present study, we demonstrated that ZEB-1 also inhibits SEMA3F in lung cancer cells. Levels of ZEB-1, but not ZEB-2, Snail or Slug, significantly correlate with SEMA3F inhibition, and overexpression or inhibition of ZEB-1 correspondingly affected SEMA3F expression. Four conserved E-box sites were identified in the SEMA3F gene. Direct ZEB-1 binding was confirmed by chromatin immunoprecipitation assays for two of these, and ZEB-1 binding was reduced when cells were treated with a histone deacetylase inhibitor. These results demonstrate that ZEB-1 directly inhibits SEMA3F expression in lung cancer cells. SEMA3F loss was associated with changes in cell signaling: increased phospho-AKT in normoxia and increase of hypoxia-induced factor 1alpha protein in hypoxia. Moreover, exogenous addition of SEMA3F could modulate ZEB-1-induced angiogenesis in a chorioallantoic membrane assay. Together, these data provide further support for the importance of SEMA3F and ZEB-1 in lung cancer progression.

Semaphorins are a large family of secreted, transmembrane and GPI-linked proteins initially characterized in the development of the nervous system and axonal guidance. Semaphorins are expressed in many tissues where they regulate normal development, organ morphogenesis, immunity and angiogenesis. They affect the cytoskeleton, actin filament organization, microtubules and cell adhesion. Semaphorin signaling is transduced by plexins, which in the case of most class-3 semaphorins requires high affinity neuropilin receptors. The neuropilins also function as receptors for VEGF and other growth factors, and their expression is often abnormal in tumors. In cancer, semaphorins have both tumor suppressor and tumor promoting functions. We review here the current status of semaphorins and their receptors in tumor development with a focus on lung cancer.