Background
Heme oxygenases are the rate-limiting enzymes in heme degradation that catalyze the conversion of heme into carbon monoxide, iron, and biliverdin. Heme oxygenase 1 (HO-1) has (cyto)protective properties and antiinflammatory, antiapoptotic, and antiproliferative capacities of HO-1 have been described in several cell types [
1,
2]. Under normal physiologic conditions HO-1 expression is low but can be upregulated in response to a wide range of stimuli and activated signaling molecules, including the HO-1 substrate heme, reactive oxygen species (ROS), nitric oxide species, prostaglandins, cytokines, growth factors such as insulin, and lipopolysaccharide [
2]. Since heat shock (and other cellular stressors) lead to upregulation of HO-1, this molecule has also been termed heat-shock protein 32 (Hsp32).
A relation between malignant behavior and alterations in expression of HO-1 may exist. Elevated HO-1 has been detected in several cancer cell lines [
3‐
6] and tumors (including lymphosarcoma, adenocarcinoma, hepatoma, glioblastoma, melanoma, prostate cancers, Kaposi sarcoma, squamous carcinoma, pancreatic cancer, brain tumors and myeloid leukemias; reviewed in [
7]), thereby affecting tumor cell apotosis, proliferation, invasion and metastasis [
7]. Furthermore, HO-1 gene polymorphisms have been associated with increased cancer susceptibility [
8,
9].
Cell adhesion is an important determinant of organised growth and the maintenance of architectural integrity. Changes in cell-cell and cell-extracellular matrix (ECM) adhesion accompany the transition from benign tumours to invasive, malignant cancers and the subsequent metastatic dissemination of tumour cells [
6,
10,
11]. Specifically, alterations in ECM remodeling have been shown to affect adhesion properties of neoplastic cells. Although several studies have linked expression of HO-1 with various stages of tumor progression [
12‐
15], the molecular mechanisms underlying HO-1-mediated changes in adhesion of neoplastic cells remain elusive.
We used gene expression profiling as a global assay to identify a common gene set directly linked to HO-1 in 14 cancer types. One of the genes that emerged was PXDN, the human homologue of the
Drosophila gene peroxidasin. PXDN is a cell surface peroxidase associated with the extracellular matrix [
12] and was found to play a key role in HO-1-dependent cell adhesion of neoplastic cells in our investigations. Our results reflect, for the first time, that HO-1 mediates genome-wide effects on transcriptional regulation of genes potentially involved in tumorigenesis. Moreover, our findings provide insights into the mechanisms underlying HO-1-dependent tumor invasion and support the notion that HO-1 represents a molecular target in cancer.
Discussion
In cancer, HO-1 influences tumor cell survival, apoptosis, invasion and metastasis as well as resistance of certain tumors to chemotherapeutic agents [
7,
17]. These changes suggest alterations of signal transduction and transcription pathways, which HO-1 affects either directly or indirectly. To identify these regulatory mechanisms and to determine the identity of the universal genes, expression of which is affected by HO-1, we silenced HO-1 expression in BeWo choriocarcinoma cells ('miHO-1') and performed gene expression profiling of these cells relative to BeWo cells which express HO-1 endogenously ('LMP'). An interesting aspect of the 214 identified genes whose expression was affected by HO-1, was the regulation of multiple genes linked to cell plasticity/motility and ECM maintenance. In the course of invasion tumor cells leave normal structures by passing through basal membrane and migrate into the surrounding stroma. These events include significant changes in cell morphology as well as close interaction of cells with extracellular matrix (ECM) and structural rearrangement of the latter. Further evidence for a role of HO-1 in modulating cell plasticity was revealed by pathway prediction analysis, which demonstrated modulation of genes of the extracellular region as well as underlying signal transduction pathways (GSEA; Fig
1). Consistent with our data, TGFB1 was identified as a HO-1 target gene in a microarray comparison of prostate cancer cells with varying HO-1 protein levels [
12].). Several potential mechanisms underlying gene regulation by HO-1 can be envisioned that also emphasize a potential role of the enzymatic products of HO-1: regulation of signaling pathways including ERK and p38 MAPK [
13], Akt/Protein kinase B [
5], and transcription factors such as AP-1, AP-2, Brn-3 [
29], PPARγ [
25], NF-kappaB [
30], HSF-1 [
31] and HIF1α [
32]. Heme containing (and carbon monoxide) responsive transcription factors such as NPAS2 [
33] and REV-ERBα/REV-ERBβ [
34,
35] modulate gene expression in response to the HO-1 enzymatic product carbon monoxide. Recent studies revealed the nuclear localization of HO-1, pointing to its role as a potential transcription factor or coregulator [
29,
36] Of note, we detected a fraction of total cellular HO-1 protein in the nucleus of BeWo cells (data not shown). Further studies are warranted to investigate potential signaling pathways triggered by HO-1, (including the role of nuclear HO-1) in gene regulation.
To provide unbiased proof for the role of HO-1 in genome-wide transcriptional regulation, irrespective of the cancer tissue type, we performed a metaprofiling analysis using the GCM database of 190 human tumors of 14 different types. The motivation of this data mining strategy was to identify which genes from the 214 putative HO-1 target genes, determined in BeWo cells, most closely correlated with the expression of HO-1 in 190 tumor samples. This unbiased comparative analysis revealed 14 HO-1 universal target genes: proteolytic ADAM8 and MMP2, acyltransferase AGPAT2, cell surface protein MICB, extracellular glycosylase ST3GAL2, amino acid transporter SLC7A1, steroid dehydrogenase HSD17B1, thiol reductase IFI30, alkaline phosphatase ALPPLA2, intracellular adapter protein CRIP2, exracellular matrix constituents BGN and COL21A1, multifunctional cytokine TGFB1, and peroxidase PXDN. The expression of these genes is strongly correlated with that of HO-1 (P = 0.00002). The results of our data mining and our subsequent statistical analyses were validated by using qRT-PCR, Western blotting, and immunostaining of LMP and miHO1 cells. Immunofluorescence staining of first trimester placenta specimens confirmed that HO-1 immunoreactivity is coupled to that of PXDN in trophoblast cells (Fig.
4), which share the capacity to migrate and invade surrounding tissues similar to malignant cells [
37]. Based on these results, we suggest that HO-1 stimulates multiple transcriptional changes and affects several cellular pathways, including extracellular matrix organization (MMP2, ADAM8, TGFβ1, BGN, COL21A1, PXDN), signaling (CRIP2, MICB), amino acid transport and glycosylation (SLC7A1 and ST3GAL2), estrogen and phospholipid biosynthesis (AGPAT2 and HSD17B1), protein stabilization (IFI30) and phosphorylation (ALPPL2). Many of these genes are directly associated with cancer; further studies are warranted to identify the role of the HO-1 associated genes in the tumorigenic proteries of HO-1.
Given that cell adhesion is intrically linked to tumor progression/invasion, and that the HO-1 gene signature features many regulators of cell adhesion, we investigated potential effects of HO-1 on cell adhesion in HO-1 silenced BeWo cells and HO-1 overexpressing 607B melanoma cells. Knockdown of HO-1 in BeWo cells reduced adhesion to various ECM molecules, having strongest effect on Laminin (Fig.
5). Stronger adhesion of 607B cells overexpressing HO-1 confirmed a positive role of HO-1 in cell adhesion (Fig.
7). Previously, we have shown that loss of HO-1 expression in BeWo cells resulted in increased cell motility, based on boyden chamber assays [
25]. Thus, at least in BeWo cells, knockdown of HO-1 decreases cell adhesion with a concomitant increase in cell motility. A reduction in cell adhesion with a concomitant increase in cell motility is one hallmark of mesenchymal-amoeboid transition (MAT), a process describing a change in (cancer cell) movement from mesenchymal to amoeboid mode. Such type of movement was shown to be characteristic of certain malignancies, including prostate cancer [
10,
11].
We hypothesized that one of the HO-1 signature genes, many of which represent potential regulators of cell plasticity, mediates the adhesion-promoting effect of HO-1. One promising and novel candidate was PXDN, which could alter cell-ECM interaction either by stabilization of the ECM through protein-protein interactions via leucine-rich repeats and immunoglobulin loops, as well as by enzymatically formed tyrosine-tyrosine crosslinks [
38]. PXDN, also known as MG50, is a peroxidase associated with the endoplasmatic reticulum, and expressed in melanoma, breast cancer, colon cancer, ovarian cancer, renal carcinoma as well as metastatic gliomas [
4,
38‐
40] Silencing of PXDN abolished the adhesion-promoting effect of endogenous HO-1 in BeWo (LMP) and 607B (MSCV-HO1) cells (Fig.
5 and Fig.
6), while PXDN knockdown did not affect cell adhesion in HO-1 deficient cells. We hypothesize that the PXDN dosage may be very critical for the adhesive response, as PXDN levels in miHO-1 cells treated with a PXDN specific siRNA were ~50 times lower compared to LMP cells (Fig.
5): If inhibition of BeWo cell adhesion correlates with PXDN - levels, maybe there exists a threshold level for PXDN. However, the phenotype of miHO1 cells could be rescued by PXDN overexpression (Fig.
6G). The reduced (~50%) matrigel invasion of PXDN-silended BeWo cells is most likely due to pro-proliferative properties of PXDN (Fig
7). However, additional mechanisms must prevail as cell growth in PXDN silenced cells was inhibited by approximately 30% after 24 hrs, the duration of the cell invasion assay. Importantly, to our knowledge, this is the first time showing functional effects of PXDN expression levels on cell adhesion and invasion. Further extensive experiments are needed to determine the molecular mechanism by which PXDN modulates cell adhesion and invasion, and how it is linked to the adhesion-promoting properties of HO-1.
To conclude, our unbiased large scale genome-wide studies clarified, for the first time, the molecular signature of HO-1 in cancer and identified the genes which are functionally, universally, and most consistently linked with HO-1 expression among multiple tumor types. The identification of the HO-1 target genes will undoubtedly help to understand the complex network of cellular and molecular events, which are linked to the role of HO-1 in cancer. Ongoing studies will shed light on the functional significance of these individual genes.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ST carried out the GeneChip and bioinformatic as well as statistical analysis and drafted the manuscript. AJ carried out adhesion assays and western blotting. SH and MK carried out the immunostaining. MM designed primers and performed real-time PCR measurements. JH generated retroviral constructs, conducted retroviral gene transductions and cell proliferation assays. JL performed cell invasion assays and transient transfections. HP and OW participated in the design and coordination of the study. MB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.