Hypoxia-inducible factors (HIFs) induced by reduced O₂ availability activate the transcription of target genes encoding proteins that play important roles in communication between cancer and stromal cells. Cancer cells were incubated under hypoxic conditions: H1299, A549 (NSCLC); Hep3B, HepG2 (HCC); HCT116, CT26 (Colon cancer); MCF-7, MDAMB231 (Breast cancer); MKN1, MKN5 (Gastric cancer); U87MG, SHSY5Y (Brain cancer); and SKOV3, SNU840 (Ovary cancer). All cells expressed HIF-1α and HIF-2α mRNA and proteins. However, cell proliferation of NSCLC, breast, gastric, and brain cancer cells under hypoxia was more dependent on HIF-1α except for HCC cells where it was more dependent on HIF-2α. Among HIF-1α dependent cells H1299 was the most affected in terms of cell proliferation by HIF-1α knockdown. To examine which cytokines are secreted in NSCLC cells by HIF-1α to communicate with stromal cells, we performed a cytokine-profiling array with H1299. We screened the top 14 cytokines which were dependent on the HIF-1α expression pattern. Among them, midkine (MDK) expression was affected the most in response to HIF-1α. MDK is a heparin-binding growth factor that promotes angiogenesis and carcinogenesis. Indeed, MDK significantly increased HUVEV endothelial cell migration and neo- vascularization in chick chorioallantoic membrane assay (CAM) assay via paracrine signaling. In addition, MDK secreted from NSCLC cells interacted with Notch2 which activated the Notch signaling pathway and induced EMT, upregulated NF-κB, and increased cancer promotion. However, in response to MDK knock down, siRNA or the MDK inhibitor, iMDK treatment not only decreased MDK-induced migration and angiogenesis of endothelial cells but also abrogated the progression and metastasis of NSCLC cells in in vitro and in vivo orthotopic and spontaneous lung metastasis models. Consequently, iMDK treatment significantly increased mice survival rates compared with the control or MDK expression group. MDK plays a very important role in the progression and metastasis of NSCLC cells. Moreover, the MDK targeting strategy provides a potential therapeutic target for the treatment of MDK-expressing lung cancers.

We have previously demonstrated that 40%‐70% of elf^+/− mice spontaneously develop hepatocellular cancer (HCC) within 15 months, revealing the importance of the transforming growth factor‐beta (TGF‐β) signaling pathway in suppressing tumorigenesis in the liver. The current study was carried out to investigate mechanisms by which embryonic liver fodrin (ELF), a crucial Smad3/4 adaptor, suppresses liver tumor formation. Histological analysis of hyperplastic liver tissues from elf^+/− mice revealed abundant newly formed vascular structures, suggesting aberrant angiogenesis with loss of ELF function. In addition, elf^+/− mice displayed an expansion of endothelial progenitor cells. Ectopic ELF expression in fetal bovine heart endothelial (FBHE) cells resulted in cell cycle arrest and apoptosis. Further analysis of developing yolk sacs of elf^−/− mice revealed a failure of normal vasculature and significantly decreased endothelial cell differentiation with embryonic lethality. Immunohistochemical analysis of hepatocellular cancer (HCC) from the elf^+/− mice revealed an abnormal angiogenic profile, suggesting the role of ELF as an angiogenic regulator in suppressing HCC. Lastly, acute small interfering RNA (siRNA) inhibition of ELF raised retinoblastoma protein (pRb) levels nearly fourfold in HepG2 cells (a hepatocellular carcinoma cell line) as well as in cow pulmonary artery endothelial (CPAE) cells, respectively. Conclusion: Taken together these results, ELF, a TGF‐β adaptor and signaling molecule, functions as a critical adaptor protein in TGF‐β modulation of angiogenesis as well as cell cycle progression. Loss of ELF in the liver leads the cancer formation by deregulated hepatocyte proliferation and stimulation of angiogenesis in early cancers. Our studies propose that ELF is potentially a powerful target for mimetics enhancing the TGF‐β pathway tumor suppression of HCC. (HEPATOLOGY 2008.)

Transforming growth factor beta (TGF-β) is an important regulator of cell growth, and loss of TGF-β signaling is a hallmark of carcinogenesis. The Smad3/4 adaptor protein β2-spectrin (β2SP) is emerging as a potent regulator of tumorigenesis through its ability to modulate the tumor suppressor function of TGF-β. However, to date the role of the TGF-β signaling pathway at specific stages of the development of hepatocellular carcinoma (HCC), particularly in relation to the activation of other oncogenic pathways, remains poorly delineated. Here we identify a mechanism by which β2SP, a crucial Smad3 adaptor, modulates cyclin dependent kinase 4 (CDK4), cell cycle progression, and suppression of HCC. Increased expression of β2SP inhibits phosphorylation of the retinoblastoma gene product (Rb) and markedly reduces CDK4 expression to a far greater extent than other CDKs and cyclins. Furthermore, suppression of CDK4 by β2SP efficiently restores Rb hypophosphorylation and cell cycle arrest in G1. We further demonstrate that β2SP interacts with CDK4 and Smad3 in a competitive and TGF-β-dependent manner. In addition, haploinsufficiency of cdk4 in β2sp+/− mice results in a dramatic decline in HCC formation compared to that observed in β2spZ+/− mice. Conclusion : β2SP deficiency leads to CDK4 activation and contributes to dysregulation of the cell cycle, cellular proliferation, oncogene overexpression, and the formation of HCCs. Our data highlight CDK4 as an attractive target for the pharmacologic inhibition of HCC and demonstrate the importance of β2sp+/− mice as a model of preclinical efficacy in the treatment of HCC. (Hepatology 2011;)

We report proteogenomic analysis of locally advanced cervical cancer (LACC). Exome-seq data revealed predominant alterations of keratinization-TP53 regulation and O-glycosylation-TP53 regulation axes in squamous and adeno-LACC, respectively, compared to in early-stage cervical cancer. Integrated clustering of mRNA, protein, and phosphorylation data identified six subtypes (Sub1-6) of LACC among which Sub3, 5, and 6 showed the treatment-resistant nature with poor local recurrence-free survival. Elevated immune and extracellular matrix (ECM) activation mediated by activated stroma (PDGFD and CXCL1high fibroblasts) characterized the immune-hot Sub3 enriched with MUC5AChigh epithelial cells (ECs). Increased epithelial-mesenchymal-transition (EMT) and ECM remodeling characterized the immune-cold squamous Sub5 enriched with PGK1 and CXCL10high ECs. We further demonstrated that CIC mutations could trigger EMT activation by upregulating ETV4, and the elevation of the immune checkpoint PVR and neutrophil-like myeloid-derived suppressive cells (FCN1 and FCGR3Bhigh macrophages) could cause suppression of T-cell activation in Sub5. Increased O-linked glycosylation of mucin characterized adeno-LACC Sub6 enriched with MUC5AChigh ECs. These results provide a battery of somatic mutations, cellular pathways, and cellular players that can be used to predict treatment-resistant LACC subtypes and can serve as potential therapeutic targets for these LACC subtypes.

To evaluate the role of risk-adapted proton beam therapy (PBT) in hepatocellular carcinoma (HCC) patients, a total of 243 HCC patients receiving risk-adapted PBT with three dose-fractionation regimens (regimen A [n = 40], B [n = 60], and C [n = 143]) according to the proximity of their gastrointestinal organs (<1 cm, 1–1.9 cm, and ≥2 cm, respectively) were reviewed: The prescribed doses to planning target volume 1 (PTV1) were 50 gray equivalents (GyE) (EQD2 [equivalent dose in 2 Gy fractions], 62.5 GyE₁₀), 60 GyE (EQD2, 80 GyE₁₀), and 66 GyE (EQD2, 91.3 GyE₁₀) in 10 fractions, respectively, and those of PTV2 were 30 GyE (EQD2, 32.5 GyE₁₀) in 10 fractions. In all patients, the five-year local recurrence-free survival (LRFS) and overall survival (OS) rates were 87.5% and 48.1%, respectively, with grade ≥3 toxicity of 0.4%. In regimens A, B, and C, the five-year LRFS and OS rates were 54.6%, 94.7%, and 92.4% (p < 0.001), and 16.7%, 39.2%, and 67.9% (p < 0.001), respectively. The five-year OS rates of the patients with the Modified Union for International Cancer Control (mUICC) stages I, II, III, and IVA and Barcelona Clinic Liver Cancer (BCLC) stages A, B, and C were 69.2%, 65.4%, 43.8%, and 26.6% (p < 0.001), respectively, and 65.1%, 40%, and 32.2% (p < 0.001), respectively. PBT could achieve promising long-term tumor control and have a potential role as a complementary or alternative therapeutic option across all stages of HCC.

β2-Spectrin is an actin-binding protein that plays an important role in membrane integrity and the transforming growth factor (TGF)-β signalling pathway as an adaptor for Smads. Loss of β2-spectrin in mice (Spnb2^−/−) results in embryonic lethality with gastrointestinal, liver, neural, and heart abnormalities that are similar to those in Smad2^+/−Smad3^+/− mice. However, to date, the role of β2-spectrin in embryogenesis, particularly in heart development, has been poorly delineated. Here, we demonstrated that β2-spectrin is required for the survival and differentiation of cardiomyocytes, and its loss resulted in defects in heart development with failure of ventricular wall thickening. Disruption of β2-spectrin in primary muscle cells not only inhibited TGF-β/Smad signalling, but also reduced the expression of the cardiomyocyte differentiation markers Nkx2.5, dystrophin, and α-smooth muscle actin (α-SMA). Furthermore, cytoskeletal networks of dystrophin, F-actin, and α-SMA in cardiomyocytes were disorganized upon loss of β2-spectrin. In addition, deletion of β2-spectrin in mice (Spnb2^tm1a/tm1a) prevented proper development of the heart in association with disintegration of dystrophin structure and markedly reduced survival. These data suggest that β2-spectrin deficiency leads to inactivation of TGF-β/Smad signalling and contributes to dysregulation of the cell cycle, proliferation, differentiation, and the cytoskeletal network, and it leads to defective heart development. Our data demonstrate that β2-spectrin is required for proper development of the heart and that disruption of β2-spectrin is a potential underlying cause of congenital heart defects.