Bortezomib induces heme oxygenase-1 expression in multiple myeloma

Abstract

Multiple myeloma (MM) is a progressive malignant disorder characterized by accumulation of plasma cells in the bone marrow. MM remains an incurable disease with a 5-y survival rate of approximately 40%. While clinical response rates to first line chemotherapeutics are high, disease relapse is inevitable, and occurs because a small fraction of the original myeloma cells appear to be resistant to treatment. Heme oxygenase-1 (HO-1) is an Nrf2 transcription factor-regulated gene that is commonly induced following oxidative stress and cellular injury, functioning to decrease oxidative stress and inflammatory responses, protecting against apoptosis and altering the cell cycle. We and others have highlighted the role of HO-1 in providing cellular protection against chemotherapeutic drugs in a number of cancer cells, which we have highlighted here in this Extra View. Furthermore, we explored the expression of HO-1 in multiple myeloma cells in response to the key anti-myeloma drugs bortezomib and lenalidomide. We show here for the first time that bortezomib increases HO-1 expression in a time- and concentration-dependent manner. Moreover, we also observe that HO-1 is increased in lenalidomide-resistant MM cell lines. Altogether, we highlight a possible role for HO-1 in basal and acquired chemoresistance in MM.

Keywords: :

• Nrf2
• bortezomib
• heme oxygenase-1
• multiple myeloma

Introduction

Multiple myeloma (MM) is a malignant neoplasm of plasma cells and represents about 10% of hematologic malignancies and 1% of all cancers. Across Europe, there are approximately 21,000 new patients per year diagnosed with MM and 16,000 deaths per year from the disease.¹ A number of significant therapeutic advances have been made in the past 15 years, including the proteasome inhibitor bortezomib as well as thalidomide and its derivative lenalidomide, which have been associated with improved survival outcomes for patients with MM.^2,3 However, despite these developments in treatment, relapse is still inevitable, and MM remains an incurable disease with a 5-y survival of about 40%. As such, considerable effort is currently being invested in developing a better understanding of myeloma biology with a view to overcoming chemotherapy failure with the ultimate goal of achieving strategies that will reliably cure MM.

One of the main characteristics of cancer cells, when compared with their parental normal counterparts, is a continuous pro-oxidant state that can lead to intrinsic oxidative stress. For instance, primary chronic lymphocytic leukemia cells have been shown to have increased reactive oxygen species (ROS) production when compared with normal lymphocytes.⁴ This persistent ROS stress may induce adaptive stress responses, allowing cancer cells to survive with elevated levels of ROS and preserve cellular viability. This aberrantly activated intracellular ROS-scavenging system could have detrimental effects on anticancer drugs that work through accumulation of ROS to stimulate cytotoxicity and cell death.⁵

In this respect, heme oxygenase-1 (HO-1) is an enzyme that has recently come to light as playing a potentially central role in cancer cell survival. HO-1 is part of a family of heme oxygenases (HO), which are enzymes that catalyze the initial rate-limiting step of heme degradation to generate biliverdin, free heme iron and carbon monoxide. The classical physiological functions of HO are to decrease oxidative stress and inflammatory responses and to protect against apoptosis by the removal of heme, a potent pro-oxidant and pro-inflammatory mediator. Two major isoforms of HO have been identified in human: an inducible isoform, HO-1 (also named heat shock protein 32), and a constitutively expressed isoform, HO-2. HO-1 is regulated by the Nrf2/antioxidant response element (ARE) located in the upstream promoter region of HO-1, and its mRNA and protein expression are commonly upregulated following oxidative stress and cellular injury.^6-8 Other mechanisms of HO-1 transcriptional regulation have also been described through the hemoprotein Bach1, a heme-sensing protein that binds to and inhibits Maf proteins, the crucial heterodimer partners that are necessary for Nrf2 to bind to ARE.^9-11 In addition, we and others have shown that NFκB and AP-1 can also regulate HO-1 transcription.^12,13 Moreover, Kirino and colleagues have shown that the pro-inflammatory cytokine tumor necrosis factorα (TNF) can induce downregulation of HO-1 in human monocytes by promoting the degradation of HO-1 mRNA.¹⁴

Taken together, we now increasingly understand that while HO-1 is physiologically regulated by multiple mechanisms, there are a variety of cancer cell types which have hijacked the HO-1 regulatory pathways to utilize this protein to protect against chemotherapy-induced increases in ROS and, thus, to promote tumor survival.

This review will highlight the major findings from studies of HO-1 in cancer and discuss their significance in the context of known HO-1 activation pathways. In addition to reviewing recent work exploring the role of HO-1 in chemotherapy resistance, we will discuss novel insights into the role of HO-1 induction in multiple myeloma cells and implications for future therapies.

HO-1 in Hematological Malignancies

Aberrant expression of HO-1 in human cancers is implicated in oncogenesis and chemoresistance.^15-17Table 1 highlights the major studies showing the effect of HO-1 in hematologic malignancies.

Table 1. HO-1 functional expression in hematological malignancies.

Leukemia	Patients/cell lines	Comments	Ref.
CML	Primary chronic phase (CP); accelerated phase (AP); and blast phase (BP) cells.	CML expressed HO-1 in a BCR/ABL-dependent manner promoting cell survival. HO-1 expression increased from CP to BP.	18, 28
CML	K562 cells expressing either wt BCR/ABL or imatinib-resistant mutants of BCR/ABL.	HO-1 targeting drugs suppressed the growth of imatinib-resistant BCR/ABL-transformed leukemic cells.	20
CLL	Primary CLL.	Nrf2 and HO-1 protein expression were higher in CLL compared with normal PBMC.	26
AML	Primary AML cells and AML cell lines.	TNF treatment together with NFκB inhibition induced HO-1 expression in AML cells protecting them from TNF-induced cell death.	13, 21
AML	Primary AML cells and AML cell lines.	Bortezomib promotes the induction of Nrf2-regulated genes: HO-1, ferritin, GSR, GCLM and NQO1 protecting AML from apoptosis.	5
AML	Primary AML cells and AML cell lines.	HO-1 expression was increased by cytarabine and daunorubicin treatment and silencing HO-1 promoted the apoptotic effect in response to these drugs.	17
MM	OPM2, IM9 and RPMI8226 myeloma cell lines.	MM cells treated with arsenic trioxide upregulated the expression of HO-1. Inhibition of this enzyme increased ROS levels and decreased MM cell proliferation.	29
MCL and MM	B lymphocytes, mantle B cell lymphoma and RPMI8226.	Malignant B cells have a greater capacity to increase HO-1 levels in response to 15d-PGJ2.	30

Work performed in chronic myeloid leukemia (CML) has shown that primary CML cells and the CML-derived K562 cell line constitutively expresses HO-1, and, furthermore, HO-1 has been identified as a BCR/ABL oncoprotein-dependent survival factor.¹⁸ Moreover, numerous studies have revealed that HO-1 provides resistance to imatinib, which inhibits the tyrosine kinase BCR-ABL, and that HO-1 silencing returns the cells to the imatinib-sensitive phenotype.^19,20

Our own investigations have shown that in AML-derived cells, but not primary cells, HO-1 is upregulated in response to TNF stimulation in conjunction with NFκB inhibition.²¹ Furthermore, this induction of HO-1 protects AML cells from cell death signals. The signaling machinery involved in regulating this response is Nrf2. HO-1 and Nrf2 may protect against the detrimental effects of inflammation and oxidative stress but may also help protect cancerous AML cells from TNF-mediated cell death, resulting in potentially clinically significant levels of apoptosis resistance. Moreover, our work and a study by Ishigatsubo and colleagues have revealed that Nrf2 is constitutively active in some AML samples but not in others.^5,22 This heterogeneity in the levels of Nrf2 observed in AML samples could be the result of acquired somatic mutations in Keap1 or Nrf2 or as a consequence of epigenetic changes that can deregulate the expression of these genes.^23,24 In addition, the study by Miyazaki et al. showed that constitutive Nrf2 induces HO-1, which provides protection for AML cells from chemotherapy-induced apoptosis.²² More recently, another study has confirmed our own findings that inhibiting HO-1 activity with zinc protoporphyrin induces apoptosis in AML cells,²⁵ suggesting that induced HO-1 expression plays a protective role in this disease, and that HO-1 is a candidate drugable target.

In lymphocytic leukemias little is known about the role of HO-1, with the possible exception of one study in which HO-1 was shown to be increased in chronic lymphocytic leukemia.²⁶ To further understand the role of HO-1 in protecting MM cells from apoptosis we describe here the expression profile of HO-1 in MM primary cells and cell lines in response to lenalidomide and bortezomib over a short (4 and 8 h) and long (12 weeks) treatment period.

Multiple Myeloma and HO-1 Chemotherapy-Induced Expression

To date, the role of HO-1 in MM has not been examined with regards to current chemotherapies. Here, we show for the first time that the clinically used MM chemotherapeutic drug bortezomib, but not lenalidomide, induces HO-1 expression over an 8-h time course (Fig. 1A). Our data show that HO-1 is upregulated in the MM primary cells used and cell lines tested, with the exception of the U266 cell line in which HO-1 was undetected. In addition, the dose-dependent nature of these drugs on HO-1 expression in RPMI8226 cells are presented (Fig. 1B).

Figure 1. Bortezomib induces HO-1 mRNA expression in MM. (A) MM primary cells and cell lines were treated with bortezomib and lenalidamide for 4 and 8 h and then total RNA was extracted. RNA was reverse transcribed and relative expression of HO-1 mRNA was measured by real-time PCR. (B) RPMI8226 cells were treated for 8 h with various doses of bortezomib and lenalidamide. Total RNA was extracted and reverse transcribed. Relative expression of HO-1 mRNA was measured by real-time PCR. Data represent the means ± SEM, from three separate experiments.

HO-1 overexpression has also been described in relapse or recurrent malignancies.²⁷ To explore the molecular basis of acquired resistance to bortezomib and lenalidomide, MM cell lines RPMI8226 and MM1S cells were exposed in vitro over a period of 3 mo to step-wise increasing concentrations of bortezomib (1 nM to 50 nM) and lenalidomide (500 nM to 20 μM). RPMI8226 and MM1S cells grown in the presence of 20 and 50 nM bortezomib (Bz20 and Bz50), 10 and 20 μM lenalidomide (Len10 and Len20) were used for characterization.

Next, we wanted to determine the expression of HO-1 in bortezomib- and lenalidomide-resistant MM cells. This was done to mimic the in vivo relapse or chemotherapy-resistant nature of the disease. Figure 2 shows that in lenalidomide-resistant MM cells, HO-1 expression has increased in both RPMI8226 and MM1S when compared with non-resistant cells. In contrast, the results for the increased expression of HO-1 in bortezomib-resistant MM1S cells were not significant. However, a slight but significant increase in HO-1 was observed in bortezomib-resistant RPMI8226 cells when compared with non-resistant cells. These results suggest that MM cells are capable of increasing the levels of HO-1 in order to protect against increased ROS and, therefore, lower the damaging effect of these chemotherapies in this disease. Thus, it is not inconceivable to propose that increased HO-1 in MM provides protection against the initial hit of bortezomib treatment (Fig. 1A and B) and, in the long-term, protection against lenalidomide treatment (Fig. 2).

Figure 2. Expression of HO-1 in chemotherapy resistant MM cell lines. MM cell lines RPMI8226 and MM1S cells were treated for 3 mo with increasing doses of bortezomib (up to 50 nM) or lenalidomide (up to 20 µM). Total RNA was extracted and reverse transcribed. Relative expression of HO-1 mRNA was measured by real-time PCR. Data represent the means ± SEM, from three separate experiments.

Concluding Remarks

In conclusion, HO-1 has been implicated as a significant chemo-resistance pathway in several hematological malignancies. Myeloma can now be added to this list. Work is ongoing to establish whether these in vitro findings can be translated into drug and trial design strategies with the overall goal of achieving improved outcomes for patients with these diseases.

Abbreviations:
MM	=	multiple myeloma
HO-1	=	heme oxygenase-1
ARE	=	antioxidant responsive element
ROS	=	reactive oxygen species

Acknowledgments

This study was supported by research funding in the form of grant support from the National Institutes of Health Research (NIHR), The Big C, Association for International Cancer Research (AICR) and Leukaemia and Lymphoma Research (LLR).