Lihua Zeng

Cancer cells that survive radiation therapy acquire HIF-1 activity and translocate towards tumour blood vessels

Tumour recurrence frequently occurs after radiotherapy, but the characteristics, intratumoural localization and post-irradiation behaviour of radioresistant cancer cells remain largely unknown. Here we develop a sophisticated strategy to track the post-irradiation fate of the cells, which exist in perinecrotic regions at the time of radiation. Although the perinecrotic tumour cells are originally hypoxia-inducible factor 1 (HIF-1)-negative, they acquire HIF-1 activity after surviving radiation, which triggers their translocation towards tumour blood vessels. HIF-1 inhibitors suppress the translocation and decrease the incidence of post-irradiation tumour recurrence. For the first time, our data unveil the HIF-1-dependent cellular dynamics during post-irradiation tumour recurrence and provide a rational basis for targeting HIF-1 after radiation therapy.

Selective Killing of Hypoxia-Inducible Factor-1–Active Cells Improves Survival in a Mouse Model of Invasive and Metastatic Pancreatic Cancer

Purpose: Pancreatic cancer is characterized by intratumoral hypoxia, early and aggressive local invasion, and metastatic potential. Hypoxia-inducible factor-1 (HIF-1) is the major transcriptional activator of hypoxia-responsive genes and intratumoral hypoxia is associated with increased risk of metastasis. However, the behavior of the cells having HIF-1 activity during the malignant progression in pancreatic cancer has not been tested. Experimental Design: We orthotopically transplanted pancreatic cancer cells stably transfected with a HIF-1–dependent luciferase reporter gene and monitored HIF-1 activity in vivo in control and POP33-treated mice. POP33 is a novel prodrug, which has potential to increase caspase-3 activity and induce apoptosis in HIF-1–active/hypoxic cells. Results:In vivo optical imaging showed that HIF-1 activity proceeded along with local invasion, the peritoneal dissemination, and the liver metastasis. HIF-1–active hypoxic cells were selectively eradicated by POP33. Moreover, selective killing of HIF-1–active hypoxic cells significantly suppressed malignant progression, resulting in a significant improvement in survival rate. Conclusions: These results show that HIF-1–active cells constitute a large proportion of invading and metastatic cells and suggest that eradication of these cells may improve the outcome in advanced pancreatic cancer, a condition for which no effective therapy currently exists.

UCHL1 provides diagnostic and antimetastatic strategies due to its deubiquitinating effect on HIF-1α

Hypoxia-inducible factor 1 (HIF-1) plays a role in tumour metastases; however, the genes that activate HIF-1 and subsequently promote metastases have yet to be identified. Here we show that Ubiquitin C-terminal hydrolase-L1 (UCHL1) abrogates the von Hippel–Lindau-mediated ubiquitination of HIF-1α, the regulatory subunit of HIF-1, and consequently promotes metastasis. The aberrant overexpression of UCHL1 facilitates distant tumour metastases in a HIF-1-dependent manner in murine models of pulmonary metastasis. Meanwhile, blockade of the UCHL1–HIF-1 axis suppresses the formation of metastatic tumours. The expression levels of UCHL1 correlate with those of HIF-1α and are strongly associated with the poor prognosis of breast and lung cancer patients. These results indicate that UCHL1 promotes metastases as a deubiquitinating enzyme for HIF-1α, which justifies exploiting it as a prognostic marker and therapeutic target of cancers.

Treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the effect of radiation therapy

Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tumour radioresistance; therefore, it is recognised as an excellent target during radiation therapy. However, the inhibition of HIF-1 in unsuitable timing can suppress rather than enhance the effect of radiation therapy because its anti-angiogenic effect increases the radioresistant hypoxic fraction. In this study, we imaged changes of HIF-1 activity after treatment with radiation and/or an HIF-1 inhibitor, YC-1, and optimised their combination. Hypoxic tumour cells were reoxygenated 6 h postirradiation, leading to von Hippel-Lindau (VHL)-dependent proteolysis of HIF-1α and a resultant decrease in HIF-1 activity. The activity then increased as HIF-1α accumulated in the reoxygenated regions 24 h postirradiation. Meanwhile, YC-1 temporarily but significantly suppressed HIF-1 activity, leading to a decrease in microvessel density and an increase in tumour hypoxia. On treatment with YC-1 and then radiation, the YC-1-mediated increase in tumour hypoxia suppressed the effect of radiation therapy, whereas on treatment in the reverse order, YC-1 suppressed the postirradiation upregulation of HIF-1 activity and consequently delayed tumour growth. These results indicate that treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the therapeutic effect of radiation, and the suppression of the postirradiation upregulation of HIF-1 activity is important for the best therapeutic benefit.

TS-1 enhances the effect of radiotherapy by suppressing radiation-induced hypoxia-inducible factor-1 activation and inducing endothelial cell apoptosis.

The therapeutic effect of concurrent chemoradiotherapy with TS‐1 has been confirmed in various solid tumors; however, the detailed mechanism of action has not yet been fully elucidated. In the present study, we identified hypoxia‐inducible factor‐1 (HIF‐1) as one of the targets of TS‐1 in chemoradiotherapy. In growth delay assays using a tumor xenograft of non‐small‐cell lung carcinoma, H441, TS‐1 treatment enhanced the therapeutic effect of single γ‐ray radiotherapy (14 Gy) and significantly delayed tumor growth by 1.58‐fold compared to radiotherapy alone (P < 0.01). An optical in vivo imaging experiment using a HIF‐1‐dependent 5HRE‐luc reporter gene revealed that TS‐1 treatment suppressed radiation‐induced activation of HIF‐1 in the tumor xenografts. The suppression led to apoptosis of endothelial cells resulting in both a significant decrease in microvessel density (P < 0.05; vs radiation therapy alone) and a significant increase in apoptosis of tumor cells (P < 0.01; vs radiation therapy alone) in tumor xenografts. All of these results indicate that TS‐1 enhances radiation‐induced apoptosis of endothelial cells by suppressing HIF‐1 activity, resulting in an increase in radiosensitivity of the tumor cells. Our findings strengthen the importance of both HIF‐1 and its downstream gene, such as vascular endothelial cell growth factor, as therapeutic targets to enhance the effect of radiotherapy. (Cancer Sci 2008; 99: 2327–2335)

Hypoxia inducible factor‐1 influences sensitivity to paclitaxel of human lung cancer cell lines under normoxic conditions

Paclitaxel (PTX) is an anticancer drug that is effective against a wide range of solid tumors. The effect of PTX on two human lung cancer cell lines, PC14PE6 and NCI‐H441 cells, was examined in an orthotopically transplanted animal model with an in vivo imaging devise. Although PTX effectively suppressed tumor growth and improved survival rate in NCI‐H441, it did not influence these in PC14PE6. In vitro experiments confirmed that PC14PE6 cells are resistant to PTX under normoxic conditions and that both cell lines were resistant to PTX under hypoxic conditions. It was found that the expression level of endogenous hypoxia inducible factor (HIF)‐1α in PC14PE6 is much higher than that in NCI‐H441 cells under normoxic conditions. Furthermore, sensitivity to PTX in these cell lines was reversed when HIF‐1α expression was decreased by siRNA specific to HIF‐1α in PC14PE6 and increased by overexpression of the exogenous HIF‐1α gene in NCI‐H441. These results suggest that HIF‐1 influences the PTX sensitivity of these cells. The authors further examined β‐tubulin, a target molecule of PTX, with western blotting and immunohistochemical analysis in these cells. The expression level of β‐tubulin was comparable in these cells under both normoxic and hypoxic conditions while the distribution of β‐tubulin and cell morphology were changed according to HIF‐1α expression levels, suggesting that HIF‐1 influences the conformation and dynamics of microtubules. These data support the potential development of HIF‐1 targeted approaches in combination with PTX, where drug resistance tends to contribute to treatment failure. (Cancer Sci 2007; 98: 1394–1401)

Effect of electromagnetic pulse exposure on permeability of blood-testicle barrier in mice.

OBJECTIVE To study the effect of electromagnetic pulse (EMP) exposure on the permeability of blood-testicle barrier (BTB) in mice. METHODS Adult male BALB/c mice were exposed to EMP at 200 kV/m for 200 pulses with 2 seconds interval. The mice were injected with 2% Evans Blue solution through caudal vein at different time points after exposure, and the permeability of BTB was monitored using a fluorescence microscope. The testis sample for the transmission electron microscopy was prepared at 2 h after EMP exposure. The permeability of BTB in mice was observed by using Evans Blue tracer and lanthanum nitrate tracer. RESULTS After exposure, cloudy Evans Blue was found in the testicle convoluted seminiferous tubule of mice. Lanthanum nitrate was observed not only between testicle spermatogonia near seminiferous tubule wall and sertoli cells, but also between sertoli cells and primary spermatocyte or secondary spermatocyte. In contrast, lanthanum nitrate in control group was only found in the testicle sertoli cells between seminiferous tubule and near seminiferous tubule wall. CONCLUSION EMP exposure could increase the permeability of BTB in the mice.

Mechanisms involved in the blood–testis barrier increased permeability induced by EMP

The blood-testis barrier (BTB) plays an important role in male reproductive system. Lots of environmental stimulations can increase the permeability of BTB and then result in antisperm antibody (AsAb) generation, which is a key step in male immune infertility. Here we reported the results of male mice exposed to electromagnetic pulse (EMP) by measuring the expression of tight-junction-associated proteins (ZO-1 and Occludin), vimentin microfilaments, and transforming growth factor-beta (TGF-beta3) as well as AsAb level in serum. Male BALB/c mice were sham exposed or exposed to EMP at two different intensities (200kV/m and 400kV/m) for 200 pulses. The testes were collected at different time points after EMP exposure. Immunofluorescence histocytochemistry, western blotting, laser confocal microscopy and RT-PCR were used in this study. Compared with sham group, the expression of ZO-1 and TGF-beta3 significantly decreased accompanied with unevenly stained vimentin microfilaments and increased serum AsAb levels in EMP-exposed mice. These results suggest a potential BTB injury and immune infertility in male mice exposed to a certain intensity of EMP.

Aberrant IDH3α expression promotes malignant tumor growth by inducing HIF-1-mediated metabolic reprogramming and angiogenesis

Cancer cells gain a growth advantage through the so-called Warburg effect by shifting glucose metabolism from oxidative phosphorylation to aerobic glycolysis. Hypoxia-inducible factor 1 (HIF-1) has been suggested to function in metabolic reprogramming; however, the underlying mechanism has not been fully elucidated. We found that the aberrant expression of wild-type isocitrate dehydrogenase 3α (IDH3α), a subunit of the IDH3 heterotetramer, decreased α-ketoglutarate levels and increased the stability and transactivation activity of HIF-1α in cancer cells. The silencing of IDH3α significantly delayed tumor growth by suppressing the HIF-1-mediated Warburg effect and angiogenesis. IDH3α expression was associated with the poor postoperative overall survival of lung and breast cancer patients. These results justify the exploitation of IDH3 as a novel target for the diagnosis and treatment of cancers.

Overexpression of keratin 17 is associated with poor prognosis in epithelial ovarian cancer

The aim of this study was to investigate the association between keratin 17 (K17) expression and the clinicopathological features of patients with epithelial ovarian cancer (EOC). K17 expression was detected by real-time quantitative RT-PCR in EOC and adjacent noncancerous tissues. In addition, K17 expression was analyzed by immunohistochemistry in 104 clinicopathologically characterized EOC cases. The expression levels of K17 mRNA and protein in EOC tissues were both significantly higher than those in noncancerous tissues. In addition, positive expression of K17 correlated with the clinical stage (p = 0.001). Furthermore, Kaplan–Meier survival analysis showed that a high expression level of K17 resulted in a significantly poor prognosis of EOC patients. Multivariate analysis revealed that EOC expression level was an independent prognostic parameter for the overall survival rate of EOC patients. Our data are the first to suggest that increased K17 expression in EOC is significantly associated with aggressive progression and poor prognosis. K17 may be an important molecular marker for predicting the carcinogenesis, progression, and prognosis of EOC.