The Cellular Oxygen Sensor PHD2 in Cancer Growth

Adequate supply of oxygen is essential for the survival of multicellular organisms. However, in several conditions the supply of oxygen can be disturbed and the tissue oxygenation is compromised. This condition is termed hypoxia. Oxygen homeostasis is maintained by the regulation of both the use and delivery of oxygen through complex, sensitive and cell-type specific transcriptional responses to hypoxia. This is mainly achieved by one master regulator, a transcription factor called hypoxiainducible factor 1 (HIF-1). The amount of HIF-1 is under tight oxygen-dependent control by a family of oxygen-dependent prolyl hydroxylase domain proteins (PHDs) that function as the cellular oxygen sensors. Three family members (PHD1-3) are known to regulate HIF of which the PHD2 isoform is thought to be the main regulator of HIF-1. The supply of oxygen can be disturbed in pathophysiological conditions, such as ischemic disorders and cancer. Cancer cells in the hypoxic parts of the tumors exploit the ability of HIF-1 to turn on the mechanisms for their survival, resistance to treatment, and escape from the oxygen- and nutrient-deprived environment. In this study, the expression and regulation of PHD2 were studied in normal and cancerous tissues, and its significance in tumor growth. The results show that the expression of PHD2 is induced in hypoxic cells. It is overexpressed in head and neck squamous cell carcinomas and colon adenocarcinomas. Although PHD2 normally resides in the cytoplasm, nuclear translocation of PHD2 was also seen in a subset of tumor cells. Together with the overexpression, the nuclear localization correlated with the aggressiveness of the tumors. The nuclear localization of PHD2 caused an increase in the anchorage-independent growth of cancer cells. This study provides information on the role of PHD2, the main regulator of HIF expression, in cancer progression. This knowledge may prove to be valuable in targeting the HIF pathway in cancer treatment.Siirretty Doriast

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

Peer reviewe

Protein phosphatase 2A (PP2A) inhibitor CIP2A indicates resistance to radiotherapy in rectal cancer

Preoperative (chemo)radiotherapy, (C)RT, is an essential part of the treatment of rectal cancer patients, but tumor response to this therapy among patients is variable. Thus far, there are no clinical biomarkers that could be used to predict response to (C)RT or to stratify patients into different preoperative treatment groups according to their prognosis. Overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) has been demonstrated in several cancers and is frequently associated with reduced survival. Recently, high CIP2A expression has also been indicated to contribute to radioresistance in head and neck squamous cell carcinoma, but few studies have examined the connection between CIP2A and radiation response regarding other malignancies. We have evaluated CIP2A protein expression levels in relation to tumor regression after preoperative (C)RT and survival of rectal adenocarcinoma patients. The effects of CIP2A knockdown by siRNA on cell survival were further investigated in colorectal cancer cells exposed to radiation. Patients with low-CIP2A-expressing tumors had more frequently moderate or excellent response to long-course (C)RT than patients with high-CIP2A-expressing tumors. They also had higher 36-month disease-specific survival (DSS) rate in categorical analysis. In the multivariate analysis, low CIP2A expression level remained as an independent predictive factor for increased DSS. Suppression of CIP2A transcription by siRNA was found to sensitize colorectal cancer cells to irradiation and decrease their survival in vitro. In conclusion, these results suggest that by contributing to radiosensitivity of cancer cells, low CIP2A protein expression level associates with a favorable response to long-course (C)RT in rectal cancer patients.</p

Kaplan Meier survival curves of <i>KRAS</i> wt colorectal cancer patients treated with anti-EGFR therapy.

<p>Progression free survival (<b>a</b>) and overall survival (<b>b</b>) of the test validation cohort according to <i>EGFR</i> gene copy number. Progression free survival (<b>c</b>) and overall survival (<b>d</b>) of the combined chemorefractory patient cohort.</p

Anti-EGFR response of colorectal cancer lines with different <i>EGFR</i> GCN and <i>KRAS</i> status.

<p>(<b>a</b>) <i>EGFR</i> GCN SISH analysis of the different cell lines. (<b>b</b>) A western blot image showing the levels of EGFR protein in the different cell lines. α-tubulin was used as a control for equal loading. The cell viability of the different cell lines at varying concentrations of (<b>c</b>) cetuximab and (<b>d</b>) panitumumab. The results are given as percentage of viable cells in comparison to the non-treated cells (mean ± SE of five experiments). <b>(e)</b> Western blots showing EGFR pathway signaling molecules in the different cell lines. The cells were pretreated with the indicated amounts of cetuximab for 24 hours in medium containing 1% FBS and given egf (25 µg/ml) for 5 minutes before lysis. The indicated signaling molecules were analyzed with western blotting. GAPDH was used as a control for equal loading.</p

Characteristics of anti-EGFR treated <i>KRAS</i> wild type metastatic colorectal cancer patients.

<p>CAP, capecitabine; EGFR = epidermal growth factor receptor; IRI, irinotecan; OXA, oxaliplatin.</p><p>Original discovery patient cohort (<b>a</b>). Independent validation patient cohort (<b>b</b>). Combined chemorefractory patient cohort (<b>c</b>).</p

Progression-free survival and overall survival of anti-EGFR treated patients according to EGFR gene copy number.

<p>The hazard ratios and confidence intervals of the original discovery, validation, and combined chemorefractory patient cohorts are shown. A high <i>EGFR</i> GCN (IHC guided SISH) is associated with an improved disease outcome in all three <i>KRAS</i> wild type metastatic colorectal cancer patient cohorts treated with anti-EGFR therapy (two independent cohorts and one combined cohort of chemorefractory patients).</p

EGFR immunohistochemistry and <i>EGFR</i> silver in situ hybridization analysis in colorectal cancer.

<p>EGFR IHC shows heterogeneous staining with intensive membranous reactivity in the middle (<b>a)</b>. <i>EGFR</i> SISH from the intensively stained area showing gene clusters (<b>b</b>). <i>EGFR</i> SISH from the surrounding areas with weak or negative EGFR IHC staining shows marginally elevated or normal gene copy numbers (<b>c–d</b>).</p