Heme oxygenase 1 facilitates cell proliferation via the B-Raf-ERK signaling pathway in melanoma
Despite therapeutic advancements (e.g. B-RAF inhibitors) targeting cutaneous melanoma, many cellular processes, including inducible heme oxygenase 1 (HO-1), counteract treatments for malignancies. So there is an urgent need to find biological treatment targets, develop new therapeutic approaches and achieve longer responses. This study aimed to explore the relationship of HO-1 and B-Raf via mediating ERK1/2 signaling on cell cycle in melanoma.
Immunohistochemistry was applied to evaluate the levels of HO-1 and B-Raf expression in melanoma tissues and adjacent healthy tissues. Co-immunoprecipitation (Co-IP) assessed the interaction of HO-1 with B-Raf. Further study overexpression and knock-down of HO-1 in A375 cell lines, especially knockout HO-1 using CRISPR-Cas9, verified HO-1 regulate cell proliferation in vivo and in vitro. Finally, Western blot analysis and qRT-PCR were performed to investigate the mechanisms by which HO-1 mediates cell cycle by B-RAF-ERK1/2 signaling.
First, histology and Co-IP show that HO-1 interacts with B-Raf directly in melanoma tissue. Further study illustrated that HO-1 overexpression promotes melanoma cell proliferation while HO-1 reduction represses melanoma cell proliferation because of HO-1 affects cell cycle. Mechanistic studies revealed that HO-1 was associated with a marked activation of B-RAF-ERK1/2 signaling and led to CDK2/cyclin E activation, thereby promoting melanoma proliferation.
Our result reveals a previously unknown mechanism that the HO-1-B-RAF-ERK axis plays an important role in melanoma cell proliferation. Therapeutic target on HO-1 could be a novel method for treating melanoma.
KeywordsHO-1 B-RAF ERK1/2 Cell cycle Melanoma
Cell counting kit
Clustered Regularly Interspaced Short Palindromic Repeats (Cas9 nuclease-null)
Fluorescence-activated cell sorting
Heme oxygenase 1
HO-1 knockout cell
Mitogen-activated protein kinase
Reactive oxygen species
Severe combined immunodeficiency
Among various skin cancers, cutaneous melanoma, a malignancy originating from melanocytes, has the highest mortality. Surgery or combinational chemotherapies have been shown to confer only modest survival benefits in advanced melanoma, with a five-year survival rate below 16% in patients with metastasis . Recently, the increased knowledge about the molecular mechanisms of melanoma has revolutionized its treatment. For example, the B-RAF inhibitors vemurafenib and dabrafenib have demonstrated an improvement in both overall survival and progression-free survival . Unfortunately, relapse usually occurs within months after treatment . Thus, there is an urgent need to develop new therapeutic approaches for melanoma treatment and prolong the responses.
Ultraviolet radiation (UVR) is estimated to be responsible for the appearance of approximately 65% of all melanomas , while heme oxygenase 1 (HO-1), the inducible form of heme oxygenase, can be induced in response to diverse cellular stress signals such as UVR and reactive oxygen species (ROS) . As an enzyme with antioxidant and antiangiogenic properties, HO-1 is usually expressed at low levels in most tissues under physiological conditions but can be highly induced following exposure to diverse chemical and physical stressors . Furthermore, an increased HO-1 expression level has been reported in different cancers, including brain tumors, chronic myeloid leukemia, melanoma and lymphosarcoma, suggesting a possible contribution of HO-1 to tumorigenesis [7, 8, 9]. These data suggest that HO-1 could is a potential biological treatment target. It is known that somatic mutations in B-RAF genes are common in melanoma. Previous studies demonstrated that B-RAF is a critical gene in the development of endometriosis , and overexpression of wild-type B-Raf is one of the mechanisms underlying the constitutive activation of the MAPK pathway that stimulates the growth of malignant melanoma cells . Moreover, a previous in vitro study showed that HO-1 increasing in a subset of thyroid cancers is associated with tumor aggressiveness and BRAFV600E expression .
Cancer is considered a disease involving cell cycle disruption. In normal cells, the cell cycle progression can be constrained, allowing the cells to discontinue cellular division under certain conditions. In contrast, the cell cycle progression is unhindered in cancer cells have unhindered. G1 phase regulation is frequently impaired in cancer cells. Thus, G1-related regulatory proteins are suitable targets for therapy. Similarly, vemurafenib, a small molecule inhibitor of a driver oncogene, binds specifically to the adenosine triphosphate (ATP) pocket of activated BRAFV600E, blocks ERK1/2 activation, halts cell cycle progression at the G0/G1 phase and promotes apoptosis. Regulation of cell cycle progression is a complex process and requires the coordinated action of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDKIs). Most tumors, including melanoma, have an abnormal G1-to-S-phase transition, mainly due to the deregulated activity of cyclin E, cyclin D, CDK2 and CDK4.
While there is a clear role of activated ERK signaling in inducing cell cycle arrest and supporting cancer cell proliferation, surprisingly little is known about the potential impact of HO-1 on the ERK signaling activity in cancers bearing oncogenic B-RAF. Here, we report a key role of HO-1 in controlling the melanoma cell cycle by regulating B-Raf expression. Endogenous HO-1 and B-Raf are highly expressed in melanoma tissues, and both are colocalized in the cytoplasm of A375 cells. Depletion of HO-1 using small interfering RNA (siRNA) or the CRISPR/Cas9-based blockade of HO-1 activity further inhibited melanoma cell proliferation, both in vitro and in vivo. The cell proliferation induced by promoting HO-1 resulted in ERK1/2 activation. Moreover, blocking HO-1 also induced cell cycle arrest at G0/G1, and overexpression of B-Raf rescued the cell cycle effect of HO-1. Our studies suggest that targeting HO-1 may be therapeutically relevant to melanoma treatments.
Cell culture and treatment
All cells used in the study were cultured in DMEM (HyClone) containing 10% fetal bovine serum and maintained at 37 °C in a humidified 5% CO2 incubator. The medium was replaced every 2 days with fresh medium to maintain cell activity. For treatment, cells were seeded in a 60 mm dish. After overnight culture, cells were exposed to UVR (25 kJ/m2, 50 kJ/m2, 100 kJ/m2) or to H2O2 (40 mM) for 6 h, and the RNA and protein were extracted 4 h or 10 h, respectively, after exposure.
Tumor samples were collected from 4 consecutive patients with melanoma who underwent surgical resection at Chongqing Hospital of Traditional Chinese Medicine (Chongqing, China) between August 2016 and August 2017. Informed consent was obtained from the patients. Those patients with preoperative anticancer treatment or with evidence of other malignancies were excluded from the study. The study protocol was approved by the local Ethics Committee of Chongqing Traditional Chinese Medicine Hospital.
Analysis of cell proliferation
Cell cycle analysis was performed using fluorescence-activated cell sorting (FACS) as previously described. Additionally, clonogenic assays and the CCK-8 assay were conducted as previously described .
Generation of knockout cell lines using the CRISPR-Cas9 technique
Generation of knockout cell lines using the Cas9 technique was performed as previously described . The gDNA for targeting HO-1 with the lentiCRISPRv2 vector was designed as follows: Oligo 1, 5′-CACCGGCTGCTGACCCATGACACCA-3′; Oligo 2, 5′-AAACTGGTGTCATGGGTCAGCAGCC-3′. Protein lysates were then extracted for further analysis.
Vector construction and lentiviral transduction
The full-length coding regions of 32 kDa HO-1 were amplified by PCR and cloned into the vector pLJM1 (Addgene #19319) for expression. The specific primers were as follows: 5’-ATACCGGTCACGAACGAGCCCAGCACC-3′ (forward) and 5’-GCATGCCTGAATTCACATGGCATAAAGCC-3′ (reverse). To knock down endogenous HO-1, the plasmids were established with pLKO.1 (Addgene #8453) according to the manufacturer’s protocol. The target sequence was as follows: 5’-AATGCTGAGTTCATGAGGAAC-3′ for HO-1. Packaging of these lentiviruses was conducted as previously described .
RNA extraction and qRT-PCR
Total cellular RNA was extracted using TRIzol reagent (TaKaRa) following the manufacturer’s protocol. RNA was reversed transcribed to cDNA using SuperScriptIII (Promega). qRT-PCR was performed with products from Applied Biological Materials Inc. (Richmond, BC, Canada) on a CFX Connect™ Real-Time PCR Detection System (USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The relative expression levels of mRNA were quantified using the ΔΔCt method.
Immunofluorescence and immunohistochemistry
Immunofluorescence and immunohistochemistry staining was carried out as previously described . The antibodies were B-Raf antibody (1:100, Abcam), p-ERK antibody (1:200, Cell Signaling Technology), and HO-1 antibody (1:200, Cell Signaling Technology). Slides were observed under a Nikon E600 microscope (Tokyo, Japan) with a digital camera.
Western blot analysis
Western blotting was performed as follows: the protein lysates were collected from the cells. The tissue lysates were separated on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Bio-Rad). The antibodies used included HO-1, B-Raf, CDK2, cyclin E, phosphorylation of ERK1/2 (p-ERK) and ERK (1:1000) antibodies, while GAPDH (1:2000) was used as a loading control. The Western blot results were further analyzed using a Bio-Rad ChemiDoc™ XRS+ system.
Xenografted tumor model
All animal experiments were performed in accordance with the ethical guidelines of Laboratory Animal Welfare and Ethics Committee Of the Third Military Medical University. For the analysis of in vivo tumor growth and invasion, 3 × 107 A375 cells in 200 μl of DMEM were subcutaneously injected into each 4- to 6-week-old nude mouse (n = 5). A ruler measured the tumor size every 5 days. At 40 days or 45 days after injection, the mice were euthanized, and the tumors were surgically isolated and weighed use electronic load cell scales.
All values are expressed as the mean ± S.D. Statistical analysis of the data was performed by a two-tailed Student’s t-test (*P < 0.05; **P < 0.01).
HO-1 interacts with B-Raf directly in melanoma
HO-1 promotes melanoma cell proliferation
Suppression of HO-1 has an anti-proliferative effect in melanoma cells
To further evaluate the tumor suppressive ability of HO-1 knockout in vivo, HO-1−/− cells were injected subcutaneously into severe combined immunodeficiency (SCID) mice. The tumor volumes resulting from the HO-1−/− cells were significantly smaller than the tumor volumes resulting from the scramble control cells (Fig. 3j). The tumor growth curves for a five-day period show that the xenograft tumors of HO-1−/−cells grew significantly slower than the scramble control cells (Fig. 3j). At the endpoint of the tumor growth experiments at day 45, the tumors from each group were excised and weighed. The HO-1 knockout tumors were much lighter than the control tumors (Fig. 3k). These results suggest that the lack of HO-1 reduces the tumor growth of A375 cells in vivo and vitro.
HO-1 facilitates the progression of the cell cycle through B-Raf
Involvement of p-ERK signaling in HO-1-induced proliferation
Melanomas arise from the pigment-producing cells called melanocytes, which normally reside in the epiderma and extend their dendrites into keratinocytes for melanin transportation and other cell-cell contacts . Melanocytes also interact with skin fibroblasts and basal cells . Thus, many researchers believe that the microenvironment of melanocytes plays an important role in melanoma development . While melanocytes are located in a relatively deep layer of the epidermis, whether UVR causes melanoma has been debated for the past decades . HO-1 is a potent anti-inflammatory intracellular mediator that catalyzes the degradation of heme to iron, biliverdin, and carbon monoxide (CO) . High level of HO-1 expression is observed in many types of cells by stimuli i.e. UVR radiation . Indeed, we found significantly increased HO-1 expression in A375 cells treated with UV. Thus far, numerous data indicate that HO-1 level is high in multiple cancer cells, including sarcoma, melanoma, and pancreatic cancer cell lines . Additionally, the upregulation of HO-1 in melanoma cancer cells is accompanied by the increased production of VEGF associated with cancer development and progression . Similarly, our study showed higher HO-1 expression in skin melanoma tissues. However, studies on the functional role of HO-1 in melanoma are rarely reported, and its function with respect to tumorigenesis and development is poorly characterized. In the current study, we clarified the cause and relationship between HO-1 and B-RAF in terms of the cell proliferation in A375 melanoma cells.
B-RAF is a proto-oncogene that is also known as v-raf murine sarcoma viral homolog B1, which belongs to the Raf kinase family . B-RAF mutations are associated with numerous types of malignant tumors, where the mutants activate the MAPK signaling pathway constitutively, resulting in uncontrolled cell proliferation and survival . However, other malignant tumors, including primary uveal melanoma and uveal melanoma, demonstrate a lack of B-RAF mutations . Some researchers found no mutations in the hotspot regions of B-RAF (i.e., exon 15) in primary clear cell ovarian carcinoma . It has been suggested that B-RAF plays an important role in tumor development by binding to specific molecular signaling molecules. Previous studies demonstrated that B-RAF is a candidate gene for the development of endometriosis , and overexpression of wild-type B-Raf is one of the causes underlying the constitutive activation of the MAPK pathway that stimulates the growth of malignant melanoma cells . Another study performed by our group found that the expression of wtB-Raf was also promoted in melanoma cells (A375, with BRAFV600E mutations) after UV treatment, and the results showed that similar to HO-1, wtB-Raf maintain a high level in skin melanoma tissues. These results led us to propose that HO-1 is related to wtB-Raf in mutant melanoma cells. Our results finally revealed that HO-1 overexpression or knockdown is correlated with the wtB-RAF level in melanoma, and the connection is even more apparent in HO-1−/− cells. Furthermore, the present study found that HO-1 physically interacts with B-RAF and promotes its kinase activity in A375 cells. These results suggest that B-RAF plays a role downstream of HO-1 in promoting cancer development.
It was known that B-RAF contributes to cell growth by regulating the cell cycle , and one implication of the observations reported here is that HO-1 may be involved in regulating cell proliferation. Further research by overexpression and knockdown of HO-1 showed that HO-1 promotes cell growth in vivo and in vitro. However, it has been suggested that gene silencing by using a siRNA approach is not sufficient for an in-depth study of the molecular mechanisms of a gene [29, 30]. Furthermore, we used a CRISPR/Cas9 approach to knock out the HO-1 gene in A375 cells, this approach enabled a comprehensive analysis of the role of HO-1 and provided strong evidence that HO-1 exerts promoter effects in A375 cells. Interestingly, the HO-1−/− cells showed a typical morphological phenotype (spindle shaped to cobblestone cell shape change) that was not observed in previous experiments with si-HO-1 where the functional depletion of HO-1 might be insufficient. In addition, the number of A375 cells in S phase of the cell cycle was significantly lower after HO-1 was blocked, while HO-1 overexpression increased this number. Our study revealed molecular mechanisms in which HO-1 induces cell arrest at G0/G1 and showed that genetic depletion of HO-1 exerts antitumor effects. CDKs are protein kinases that regulate the cell cycle by binding to cyclin [31, 32]. The CDK2-cyclin E complex is a major player in cell proliferation that is required for progression through G1 phase and entry into S phase of the cell cycle . Moreover, CDK2 depletion suppresses cell cycle progression in melanoma cells . Here, we noted that knockdown and knockout of HO-1 in melanoma cells significantly downregulated both CDK2 and cyclin E protein levels, whereas HO-1 overexpression could reverse these effects on CDK2 and cyclin E. Finally, via rescue methods in HO-1−/− cells, this study provided proof that the HO-1- cells induced changes in the cell cycle through the cyclin E and CDK2 regulatory proteins were associated with B-Raf expression. The above results indicate that the HO-1-regulated wtB-RAF expression may lead to an increase in the levels of the CDK2-cyclin E complex that stimulates the G1/S transition, which is probably involved in HO-1-induced melanoma cell proliferation.
Some investigators have studied the downstream signaling pathways of B-RAF and found that the RAS/RAF/MAPK pathway is a critical step in the initiation of melanocyte neoplasias . Furthermore, the identification of factors that modulate the balance in favor of ERK may provide additional targets for effective therapy . Our data also confirmed the ERK1/2 was activated in melanoma tissue, as found in previous research . Given that the effects of B-RAF inhibition on cell viability are ERK dependent, we postulate that HO-1-promoted cell growth is mediated through ERK activation. In line with previous studies, a major study suggested the ERK1/2 kinase pathways mediated HO-1 activation and strongly supported the role of HO-1 in directly quenching oxidative stress, normalizing intracellular redox balance, and enhancing cellular survival . Interestingly, HO-1, as an upstream molecule, may promote cell cycle changes via a B-RAF-ERK1/2-dependent mechanism. In addition, suppressing ERK by PD98059 may result in a marked downregulation of CDK2/cyclin E. In contrast, CDK2/cyclin E was restored in the cells transfected with HO-1. This finding may be associated with the activation of B-RAF/ERK signaling, which rescues the G1 cell cycle arrest and promotes cell proliferation by modulating the G1/S transition while enhancing CDK2/cyclin E activities.
In conclusion, as summarized in Fig. 6i, the present study demonstrates that HO-1 promotes cell proliferation via the B-Raf-ERK signaling pathway in melanoma. HO-1 could be a potential novel anticancer target for melanoma therapy.
The authors thank Prof. Jiankang Liu (Xi’an Jiaotong University) and Jörg W. Bartsch for their helpful comments and suggestions on the manuscript.
This work was supported by the National Natural Science Foundation of China under Grant 81570703, the National Science Foundation for Young Scholars (81704090, 81503588),and the Natural Science Foundation of Chongqing (cstc2015jcyjA10113, cstc2017jcyjbx0044). Both projects are not in a financial or personal relationship to this study.
Availability of data and materials
All data generated in this study are included in the manuscript.
LL, YW and CXB performed the experiments. XYH helped with mouse breeding and flow cytometry. WQN and EWW provided specific reagents and human skin samples. LL, YW, WX, QCD designed the conceptual idea for this study. YW, WX, JLZ and MFN wrote the manuscript. All the authors approved the submission of this manuscript in its final form.
Ethics approval and consent to participate
All animal procedures were performed under the ethical guidelines of Laboratory Animal Welfare and Ethics Committee Of the Third Military Medical University and according to the recommendations of the Chongqing Experimental Animal Regulation Board (SCXK-PLA-20140011); the approval number is SYXK-PLA-20140031.
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