Hypoxia Induces the Expression of a 43-kDa Protein (PROXY-1) in Normal and Malignant Cells

doi:10.1006/bbrc.2000.3475

Biochemical and Biophysical Research Communications

Volume 276, Issue 1, 16 September 2000, Pages 321-328

https://doi.org/10.1006/bbrc.2000.3475 Get rights and content

Abstract

This study was designed to determine the expression of cellular factors that may participate in phenotypic changes that occur under conditions of hypoxia. Using the RT-PCR differential display method, we isolated a cDNA fragment corresponding to a gene whose expression was induced in trophoblast and breast carcinoma cells cultured under 1 or 2% oxygen vs 4% oxygen or higher. This gene encodes a 43-kDa protein initially identified in homocysteine-treated endothelial cells and later shown to be upregulated in various human and mouse cell types (termed RTP, Drg1, Cap43, rit42, Ndr1). Herein we refer to this gene product as PROXY-1, for $P$ rotein $R$ egulated by $OXY$ gen- $1$ . Elevated mRNA and protein levels were first observed in cells cultured in 1% oxygen for 8 h. Although PROXY-1 mRNA levels returned to near-control values within 2 h of reexposure to 20% oxygen, protein levels remained high 72 h after reexposure to 20% oxygen. Treatment of cells with hypoxia mimics such as cobalt or iron chelators also increased PROXY-1 expression. Moreover, presence of 30% carbon monoxide in the hypoxic atmosphere abrogated the upregulation of PROXY-1 expression. These findings suggest that hypoxia upregulates PROXY-1 levels through a heme protein-dependent pathway and that assessment of PROXY-1 expression may be of potential use in evaluating tissue hypoxia.

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Multiple other agents, including Ca2 + [139] and heavy metal ions (e.g., Co2 + [140], Ni2 + [42,140]), androgens [68,125], DNA-damaging agents (e.g., actinomycin D), forskolin [65], lysophosphatidylcholine [30], okadaic acid [42,141], sulfhydryl reducing agents and tunicamycin [40], ligands of the peroxisome proliferator-activated receptor (PPAR)-γ and retinoid X receptor (RXR) [25], iron chelators [96,111,142,143] and vitamins A and D3 [70,144,145] also promote NDRG1 expression [49,146]. Further, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) [23,43,147], p53 [41,79,148–150] and the von Hippel–Lindau protein (pVHL) [32] have been reported to promote NDRG1 expression. Three hypoxia-inducible factor (HIF)-1-binding sites have been identified in the non-coding sequence of NDRG1, of which one is located in the promoter region and two are located in the 3′ untranslated region [151].
N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.
NDRG1/Cap43/Drg-1 may predict tumor angiogenesis and poor outcome in patients with lung cancer
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Expression of N-myc downstream-regulated gene 1 (NDRG1)/Cap43 is a prognostic indicator of human malignancies according to the tumor type in which it occurs. We investigated how NDRG1/Cap43 could affect tumor growth and angiogenesis in non–small-cell lung cancer (NSCLC) in vivo using an animal experimental model, and also how it could affect tumor angiogenesis and prognosis in NSCLC patients.
Knockdown of NDRG1/Cap43 in lung cancer cells using a specific small interfering RNA resulted in growth rates in culture that were similar to those of counterpart control cells, but decreased tumor growth rates in vivo markedly. Stable NDRG1/Cap43 knockdown did not induce consistent changes in the expression of Epidermal growth factor receptor (EGFR) family proteins and c-Met in two human lung cancer cell lines in vitro. However, cell lines with NDRG1/Cap43 knockdown showed markedly decreased production of the potent angiogenic factors vascular endothelial growth factor-A and interleukin-8. Cells with knockdown of NDRG1/Cap43 showed marked reduction of tumor-induced angiogenesis. Using immunohistochemistry, we examined 182 surgically resected specimens of NSCLC for expression of NDRG1/Cap43 and tumor angiogenesis. High microvessel density in the tumor was significantly associated with nuclear positivity for NDRG1/Cap43 in both adenocarcinoma (p = 0.003) and squamous cell carcinoma (p=0.041). For both adenocarcinoma (p = 0.031) and squamous cell carcinoma (p=0.034), the survival curve of patients negative for nuclear NDRG1/Cap43 expression differed significantly from that of patients who were positive.
Therefore, the expression of NDRG1/Cap43 may be predictive of tumor angiogenesis and poor prognosis in NSCLC.
Metastasis suppressor genes. At the interface between the environment and tumor cell growth
2011, International Review of Cell and Molecular Biology
Citation Excerpt :
Drg1 was first identified as a tumor suppressor in human bladder and pancreatic cancers (Kurdistani et al., 1998), but overexpression in breast, colon, and prostate cancer cell lines suppressed metastasis without suppressing tumorigenicity (Guan et al., 2000), a pattern which is supported in limited clinical studies in breast, prostate, and liver cancers (Bandyopadhyay et al., 2003; Chua et al., 2007). DRG-1 appears to be downstream of many cancer- and metastasis-associated signaling pathways, such as p53 (Kim et al., 2004; Kurdistani et al., 1998), PI3K/PTEN (Bandyopadhyay et al., 2004), PKC (Fujii et al., 2008), hypoxia (Agarwala et al., 2000; Bandyopadhyay et al., 2003; Park et al., 2000), and in breast and prostate tumors, estrogens and androgens (Ulrix et al., 1999). Downstream mediators include VEGF and IL8 which are both involved in angiogenesis (Maruyama et al., 2006).
The molecular mechanisms and genetic programs required for cancer metastasis are sometimes overlapping, but components are clearly distinct from those promoting growth of a primary tumor. Every sequential, rate-limiting step in the sequence of events leading to metastasis requires coordinated expression of multiple genes, necessary signaling events, and favorable environmental conditions or the ability to escape negative selection pressures. Metastasis suppressors are molecules that inhibit the process of metastasis without preventing growth of the primary tumor. The cellular processes regulated by metastasis suppressors are diverse and function at every step in the metastatic cascade. As we gain knowledge into the molecular mechanisms of metastasis suppressors and cofactors with which they interact, we learn more about the process, including appreciation that some are potential targets for therapy of metastasis, the most lethal aspect of cancer. Until now, metastasis suppressors have been described largely by their function. With greater appreciation of their biochemical mechanisms of action, the importance of context is increasingly recognized especially since tumor cells exist in myriad microenvironments. In this chapter, we assemble the evidence that selected molecules are indeed suppressors of metastasis, collate the data defining the biochemical mechanisms of action, and glean insights regarding how metastasis suppressors regulate tumor cell communication to–from microenvironments.
NDRG1/2 expression is inhibited in primary acute myeloid leukemia
2010, Leukemia Research
Citation Excerpt :
Further studies are needed to clarify whether NDRG1 directly or indirectly enhances expression of myeloid transcription factors. Moreover, NDRG1 is upregulated under hypoxic conditions [21], and low oxygen conditions were shown to promote maturation of myeloid leukemic cells (reviewed in [22]). In addition, ATRA-induced HIF-1α protein might not only enhance neutrophil differentiation by increasing the transcriptional activity of the myeloid-associated transcription factors C/EBPA and AML1 [23], but also by inducing NDRG1 via HIF-1α.
Expression of N-myc downregulated gene 1 (NDRG1) is associated with growth arrest and differentiation of tumor cells. In hematopoietic cells, NDRG1 was identified in a screen for differentiation-related genes in human myelomonocytic leukemic U937 cells. In the present study, we found significantly higher NDRG1 mRNA levels in granulocytes of healthy donors than in primary acute myeloid leukemia (AML) cells. Another NDRG family member, NDRG2, was significantly higher expressed in normal macrophages compared to primary AML cells. Moreover, NDRG1 mRNA levels increased in two acute promyelocytic leukemia (APL) patients as well as in NB4 and HT93 APL cells upon all-trans retinoic acid (ATRA) therapy. In line with these observations, silencing of NDRG1 diminished neutrophil differentiation of leukemic cell lines. In conclusion, we found an association of low NDRG1 levels with an immature cell phenotype and provide evidence that NDRG1 is functionally involved in neutrophil maturation.
N-myc downstream regulated gene 1 (ndrg1) functions as a molecular switch for cellular adaptation to hypoxia
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¹: To whom correspondence and reprint requests should be addressed at Department of Anatomy and Cell Biology, Botterell Hall, 9th Floor, Queen's University, Kingston, Ontario, Canada K7L 3N6. Fax: (613) 545-2566. E-mail: [email protected].

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Regular ArticleHypoxia Induces the Expression of a 43-kDa Protein (PROXY-1) in Normal and Malignant Cells

Abstract

Blood

Exp. Cell. Res.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Exp. Cell Res.

J. Biol. Chem.

Placenta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Mech. Dev.

J. Biol. Chem.

J. Biol. Chem.

Int. J. Radiat. Oncol. Biol. Phys.

Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis

Nature

Exposure to hypoxia, glucose starvation and acidosis: Effect on invasive capacity of murine tumor cells and correlation with cathepsin (L + B) secretion

Clin. Exp. Metastasis

Hypoxia induces DNA overreplication and enhances metastatic potential of murine tumor cells

Proc. Natl. Acad. Sci. USA

Regular Article
Hypoxia Induces the Expression of a 43-kDa Protein (PROXY-1) in Normal and Malignant Cells