Terhi Jokilehto

Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.

Overexpression and nuclear translocation of hypoxia-inducible factor prolyl hydroxylase PHD2 in head and neck squamous cell carcinoma is associated with tumor aggressiveness.

PURPOSE: Hypoxia in tumors is associated with poor prognosis and resistance to treatment. The outcome of hypoxia is largely regulated by the hypoxia-inducible factors (HIF-1alpha and HIF-2alpha). HIFs in turn are negatively regulated by a family of prolyl hydroxylases (PHD1-3). The PHD2 isoform is the main down-regulator of HIFs in normoxia and mild hypoxia. This study was designed to analyze the correlation of the expression and subcellular localization of PHD2 with the pathologic features of human carcinomas and HIF-1alpha expression. EXPERIMENTAL DESIGN: The expression of PHD2 was studied from paraffin-embedded normal tissue (n = 21) and head and neck squamous cell carcinoma (HNSCC; n = 44) by immunohistochemistry. Further studies included PHD2 mRNA detection and HIF-1alpha immunohistochemistry from HNSCC specimens as well as PHD2 immunocytochemistry from HNSCC-derived cell lines. RESULTS: In noncancerous tissue, PHD2 is robustly expressed by endothelial cells. In epithelium, the basal proliferating layer also shows strong expression, whereas the more differentiated epithelium shows little or no PHD2 expression. In HNSCC, PHD2 shows strongly elevated expression both at the mRNA and protein level. Moreover, PHD2 expression increases in less differentiated phenotypes and partially relocalizes from the cytoplasm into the nucleus. Endogenously high nuclear PHD2 is seen in a subset of HNSCC-derived cell lines. Finally, although most of the tumor regions with high PHD2 expression show down-regulated HIF-1alpha, regions with simultaneous HIF-1alpha and PHD2 expression could be detected. CONCLUSIONS: Our results show that increased levels and nuclear translocation of the cellular oxygen sensor, PHD2, are associated with less differentiated and strongly proliferating tumors. Furthermore, they imply that even the elevated PHD2 levels are not sufficient to down-regulate HIF-1alpha in some tumors.

The role of HIF prolyl hydroxylases in tumour growth

•u2002 Hypoxia in tumours •u2002 Overview of the oxygen‐sensing mechanisms •u2002 The family of HIF hydroxylases •u2002 Expression of PHDs in normal tissues •u2002 Regulation of PHDs •u2002 PHDs in cell growth and differentiation •u2002 Expression of PHDs in cancer •u2002 Function of the PHDs in cancer •u2002 Other dioxygenase targets in cancer and future directions

Heat- and cold-inducible regulation of HSP70 expression in zebrafish ZF4 cells

Elevated temperature induces a rapid heat shock transcription factor (HSFs)-mediated expression of heat shock (hsp) genes. The effect of cold exposure on hsp gene expression has hardly been investigated, although ectothermic animals experience both cold and heat stress. We have previously shown in zebrafish that the expression of hsf1a and a unique isoform hsf1b vary in a tissue-specific manner upon heat stress. In the current study, using a zebrafish (Danio rerio) embryonic cell line (ZF4), we have compared the effects of heat shock (28-->37 degrees C) vs. cold shock (28-->20 degrees C) on the expression of ahsf1a, zhsf1b and hsp70. Concomitantly, the suitability of the ZF4 cells as a model system was verified. The expression pattern of HSP70 proteins following heat or cold exposure is distinct, and the total HSP70 level is upregulated or stable, respectively. Moreover, heat exposure specifically increases the ratio of zhsf1a/b expression (10-fold), whereas cold exposure decreases it to one half. These data suggest that the zhsf1a/zhsf1b ratio is regulated in a temperature-dependent manner, and the ratio may be indicative of the stressor-specific HSP70 expression. Furthermore, the response in ZF4 cells upon heat shock resembles the response observed in zebrafish liver and thus, supports the use of this cell line in stress response studies.

Hypoxic Conversion of SMAD7 Function from an Inhibitor into a Promoter of Cell Invasion

Smad7 is an inhibitor of the transforming growth factor-beta-activated signaling pathway. Under well-oxygenated conditions, Smad7 is a potent inhibitor of carcinoma cell invasion. Paradoxically, however, the expression of Smad7 is upregulated across several cancers and may promote cancer progression. Hypoxia, which is frequently met in solid tumors, is an enhancer of carcinoma cell invasion and cancer progression. Here, we report that hypoxia activates the expression of Smad7 in a hypoxia-inducible factor- and von Hippel-Lindau protein-dependent manner. As expected, in normoxia, the forced expression of Smad7 inhibited carcinoma cell invasion. In contrast with the normoxic condition, the inhibitory effect of Smad7 was lost under hypoxia. The block in carcinoma cell invasion by forced expression of Smad7 was released by hypoxia in two invasive carcinoma cell lines. Moreover, the noninvasive HaCaT keratinocytes become invasive upon simultaneous hypoxia and transforming growth factor-beta stimulus. The hypoxia-activated invasion was attenuated by inhibiting Smad7 expression by short interfering RNA. Finally, the increased Smad7 expression in human carcinomas correlated with hypoxic gene expression. The data provide evidence that hypoxia could convert Smad7 function from an invasion inhibitor into an activator of invasion. Furthermore, they might shed light as to why increased Smad7 expression is detected in cancers.

Prolyl hydroxylase PHD3 enhances the hypoxic survival and G1 to S transition of carcinoma cells.

Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells.

Heterogeneous EGFR Gene Copy Number Increase Is Common in Colorectal Cancer and Defines Response to Anti-EGFR Therapy

Anti-EGFR therapy is commonly used to treat colorectal cancer (CRC), although only a subset of patients benefit from the treatment. While KRAS mutation predicts non-responsiveness, positive predictive markers are not in clinical practice. We previously showed that immunohistochemistry (IHC)-guided EGFR gene copy number (GCN) analysis may identify CRC patients benefiting from anti-EGFR treatment. Here we tested the predictive value of such analysis in chemorefractory metastatic CRC, elucidated EGFR GCN heterogeneity within the tumors, and evaluated the association between EGFR GCN, KRAS status, and anti-EGFR antibody response in CRC cell lines. The chemorefractory patient cohort consisted of 54 KRAS wild-type (WT) metastatic CRC patients. EGFR GCN status was analyzed by silver in situ hybridization using a cut-off value of 4.0 EGFR gene copies/cell. KRAS-WT and KRAS mutant CRC cell lines with different EGFR GCN were used in in vitro studies. The chemorefractory CRC tumors with EGFR GCN increase (≥4.0) responded better to anti-EGFR therapy than EGFR GCN (<4.0) tumors (clinical benefit, Pu200a=u200a0.0004; PFS, HRu200a=u200a0.23, 95% CI 0.12–0.46). EGFR GCN counted using EGFR IHC guidance was significantly higher than the value from randomly selected areas verifying intratumoral EGFR GCN heterogeneity. In CRC cell lines, EGFR GCN correlated with EGFR expression. Best anti-EGFR response was seen with KRAS-WT, EGFR GCNu200a=u200a4 cells and poorest response with KRAS-WT, EGFR GCNu200a=u200a2 cells. Anti-EGFR response was associated with AKT and ERK1/2 phosphorylation, which was effectively inhibited only in cells with KRAS-WT and increased EGFR GCN. In conclusion, IHC-guided EGFR GCN is a promising predictor of anti-EGFR treatment efficacy in chemorefractory CRC.

Expression of the cellular oxygen sensor PHD2 (EGLN-1) predicts radiation sensitivity in squamous cell cancer of the head and neck

Purpose: Prolyl hydroxylase domain proteins constitute a family of oxygen sensors that regulate the expression of hypoxia-inducible factor-1 alpha (HIF-1α), which mediates transcription of many genes under low oxygen concentration. PHD2 (Prolyl hydroxylase domain protein 2) isoform is the main regulator of HIF-1α degradation in normoxia and mild hypoxia. The aim of our study was to evaluate the relationship between expression of PHD2 and radiosensitivity in squamous cell cancer of the head and neck (HNSCC). Patients and methods: Paraffin embedded sections from untreated primary tumours were obtained for immunohistochemistry in 48 HNSCC patients scheduled for preoperative radiotherapy (RT). Nuclear expression of PHD2 was evaluated as a percentage of tumour cells showing positively stained nucleus. RT was a split-course accelerated hyperfractionated regimen (1.6 Gy twice a day) in 15 patients and standard in 33 patients. Viability of cancer cells was routinely evaluated histologically from resected tumours at planned surgery 3–4 weeks after RT. The follow-up time after multimodality treatment was five years or until death. Results: PHD2 expression was low in normal tissues and well differentiated tumours. The expression was increased and predominantly nuclear in poorly differentiated tumours. In tumours later found to be sterilised by RT, nuclear PHD2 expression was markedly lower as compared to tumours showing viable cells at surgery (p = 0.04). A low nuclear staining of PHD2 (<10% of PHD2-positive nuclei) in the primary tumour was found to associate with good radiation response (p = 0.005). Conclusions: We found low PHD2 expression and in particular low nuclear expression to predict a favourable response to RT. Therefore, nuclear PHD2 expression may act as a surrogate marker for radiation resistance in HNSCC.

Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth.

Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1alpha and -2alpha). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells. The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1alpha or -2alpha expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.

EGFR gene copy number predicts response to anti-EGFR treatment in RAS wild type and RAS/BRAF/PIK3CA wild type metastatic colorectal cancer.

Anti‐EGFR antibodies are used for the treatment of RAS wild type metastatic colorectal cancer. We previously showed that EGFR gene copy number (GCN) predicts response to anti‐EGFR therapy in KRAS exon 2 wild type metastatic colorectal cancer. The aim of our study was to analyse the predictive role of EGFR GCN in RAS/BRAF/PIK3CA wild type metastatic colorectal cancer. The material included 102 patients with KRAS exon 2 wild type metastatic colorectal cancer treated with anti‐EGFRu2009±u2009cytotoxic therapy. Next generation sequencing was used for KRAS, NRAS, BRAF and PIK3CA gene mutation analyses. EGFR GCN was analysed by EGFR immunohistochemistry guided automated silver in situ hybridisation. Increased EGFR GCN (≥4.0) predicted a better response and prolonged progression free survival in anti‐EGFR treated RAS/BRAF/PIK3CA wild type patients (Log‐rank test, pu2009=u20090.0004). In contrast, survival of RAS/BRAF/PIK3CA wild type, EGFR GCN below 4.0 patients did not differ from patients with mutant RAS, BRAF or PIK3CA. Our study indicates that EGFR GCN predicts anti‐EGFR treatment efficacy in patients with RAS/BRAF/PIK3CA wt metastatic CRC. Tumours with EGFR GCN below 4.0 appear to be as refractory to anti‐EGFR treatment as tumours with mutation in any of the RAS/RAF/PIK3CA pathway genes.