Abstract
Homeodomain interacting protein kinase-2 (HIPK2) is a member of the HIPK family of stress-responsive kinases that modulates cell growth, apoptosis, proliferation and development. HIPK2 has several well-characterised tumour suppressor roles, but recent studies suggest it can also contribute to tumour progression, although the underlying mechanisms are unknown. Herein, we have identified novel crosstalk between HIPK2 and the cytoprotective transcription factor NRF2. We show that HIPK2 is a direct transcriptional target of NRF2, identifying a functional NRF2 binding site in the HIPK2 gene locus and demonstrating for the first time a transcriptional mode of regulation for this kinase. In addition, HIPK2 is required for robust NRF2 responsiveness in cells and in vivo. By using both gain-of-function and loss-of-function approaches, we demonstrate that HIPK2 can elicit a cytoprotective response in cancer cells via NRF2. Our results have uncovered a new downstream effector of HIPK2, NRF2, which is frequently activated in human tumours correlating with chemoresistance and poor prognosis. Furthermore, our results suggest that modulation of either HIPK2 levels or activity could be exploited to impair NRF2-mediated signalling in cancer cells, and thus sensitise them to chemotherapeutic drugs.
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Introduction
Homeodomain interacting protein kinase-2 (HIPK2) is a member of the HIPK family of stress-responsive kinases, and it modulates cell proliferation, differentiation, apoptosis and development.1, 2, 3, 4, 5, 6, 7 HIPK2 responds to a variety of physiological stresses,3, 8, 9, 10, 11, 12 transforming these cues into changes in transcriptional programs, which in turn enables cells to adapt to and survive the original insult. Although HIPK2 is highly regulated at the posttranslational level,9, 10, 13, 14, 15, 16, 17, 18, 19, 20 essentially no information exists about its transcriptional regulation.
HIPK2 is considered to be a potential haplo-insufficient tumour suppressor as it can promote apoptosis in response to chemotherapeutic drugs and radiation, mainly by phosphorylating p53 at S46,1, 21 which then induces expression of pro-apoptotic genes. Additionally, HIPK2 can also protect cells against genome instability induced by genotoxic agents by promoting DNA damage repair pathways.17, 22 Interestingly, accumulating evidence suggests that HIPK2 may also support tumour progression: the expression of HIPK2 is significantly higher in cervical cancer than in healthy tissue23 and in aggressive meningiomas (when compared with benign meningiomas), where it positively associates with tumour progression.24 HIPK2 is also amplified in pilocytic astrocytoma25 and in ovarian and prostate tumours (The Cancer Genome Atlas), and positively associates with cell growth in various cancer cell lines.25, 26, 27 These results imply that HIPK2 might play a dual role in cancer depending on context, either acting as a tumour suppressor or facilitating tumour progression. While the pathways involved in the tumour suppressor role of HIPK2 are relatively well understood, the underlying mechanisms mediating its cytoprotective function(s) remain unclear.
NRF2 (nuclear factor erythroid 2 (NF-E2) p45-related factor 2, encoded by NFE2L2) is the master regulator of oxidative stress responses, which allows adaptation and survival during stress conditions. NRF2 controls the expression of a battery of genes, which encode antioxidant and drug-metabolising enzymes, as well as drug transporters (for example, HO1, GSTs, NQO1 and MRPs), all of which contain antioxidant response elements (AREs) within their promoter/enhancer regions.28, 29 In normal cells, NRF2 activity is kept low under non-stress conditions by its rapid proteasomal degradation, which is principally mediated by KEAP1 (a substrate adaptor for a Cul3-based E3 ubiquitin ligase).30, 31 Upon exposure to electrophiles or reactive oxygen species, KEAP1 undergoes conformational changes that impair its substrate adaptor function, leading to the accumulation of newly synthesised NRF2, which can then translocates to the nucleus and activates its target genes.32 Furthermore, NRF2 controls the expression of a number of its own regulators (for example, KEAP1, p66, p62), thus creating autoregulatory loops that control the amplitude/duration of its own response.33, 34, 35
Although NRF2 is cytoprotective and its transient activation is linked with chemoprevention,36 it has become apparent that its sustained activation protects tumour cells against chemo- and radiotherapy and can promote metabolic activities that support cell proliferation and tumour growth.37, 38, 39, 40 Not surprisingly, therefore, NRF2 is often constitutively activated in human tumours,39, 41 where it is associated with poor prognosis.42, 43, 44 This sustained activation of NRF2 is especially relevant in lung tumours, where due to mutations in KEAP1 or NFE2L2, NRF2 is constitutively activated in 30–60% of cases.41, 45 Additionally, NRF2 can be upregulated by the oncogenic mutant KRAS, BRAF and Myc and by loss of PTEN,46, 47 suggesting that aberrant activation of NRF2 is a common event in many cancer types.
In this study, we describe for the first time the existence of crosstalk between HIPK2 and NRF2 in which NRF2 regulates HIPK2 expression, and in turn, HIPK2 positively shapes the NRF2 response.
Results and discussion
NRF2 transcriptionally regulates basal and inducible levels of HIPK2
Previously, we and others have shown that HIPK2 can affect redox balance and might regulate oxidative stress responses.10, 48, 49 As NRF2 is arguably the main transcription factor associated with oxidative stress responses, we wanted to test whether there was a link between NRF2 and HIPK2. To do so, we first used A549 cells (lung cancer cells that possess constitutively active NRF2 due to inactivating mutations on KEAP150). We found that NRF2 knockout cells (NRF2-KO) have reduced protein and messenger RNA (mRNA) basal levels of HIPK2; conversely, NRF2 reconstitution restored both the protein and the mRNA levels of HIPK2 (Figure 1a). To monitor NRF2 activity, we used the prototypic NRF2 target gene NQO1 (Figure 1a; Supplementary Figure S1A). To our knowledge, there are no studies to date addressing the transcriptional regulation of HIPK2. We therefore confirmed the effect of NRF2 on HIPK2 mRNA levels, first by using different NRF2-KO cell lines (Supplementary Figures S1B and S1C) and second, by using various short hairpin RNA (shRNAs) against NRF2 (Supplementary Figure S1D). These results demonstrate that NRF2 regulates the steady-state levels of HIPK2.
In order to answer whether NRF2 activation is also able to induce HIPK2, we used two different approaches. In a pharmacological approach, we used two classical NRF2 activators, hydrogen peroxide (H2O2) and sulforaphane (SFN), which disrupt the KEAP1-dependent NRF2 degradation. We found that NRF2 activators increase both mRNA and protein levels of HIPK2 in H1299 cells (lung cancer cells with functional KEAP1), and that this induction requires NRF2 (Figure 1b). Of note, other KEAP1-proficient cell lines showed similar behaviour (Supplementary Figure S1E), and an overexpressed flag-tagged HIPK2 construct does not get stabilised by neither of NRF2 activators (Supplementary Figure S1F). Additionally, we used a genetic model: by deleting the KEAP1-binding motif within the endogenous NRF2, we produced cells that harbour a constitutively active NRF2 gain-of-function (GOF) mutant. Such GOF mutations are often found in tumours, and have been associated with malignancy.43, 44, 51 Thus, NRF2-GOF cells provide a physiologically relevant model of sustained NRF2 activation in malignant cells. We found that H1299 NRF2-GOF cells have elevated protein and mRNA levels of HIPK2 and NQO1 when compared with their WT counterparts (Figure 1c; Supplementary Figure S1G). The upregulation of HIPK2 observed in NRF2-GOF cells is not cancer cell type-specific (Supplementary Figure 1H). This model demonstrates that sustained NRF2 activation increases HIPK2 expression.
To identify the region(s) in the HIPK2 gene that is responsible for its regulation by NRF2, we performed an in silico analysis of the HIPK2 promoter to identify potential AREs that might be bound by NRF2. The minimal proposed consensus ARE sequence is TGASnnnGC (where S=C or G).52 As active AREs are often located close to the transcription start site, we focused on two ARE sequences we identified between −1 and −2000 bp within the HIPK2 promoter, referred as ARE1 (at −246 bp: 5′-TGAGAGGGC-3′) and ARE2 (at −1794 bp: 5′-TGACTTAGC-3′). Additionally, Malhotra et al.52 using ChIP-seq in mouse cells, detected 1256 peaks as potential NRF2 binding sites. Among these, we identified a peak within a HIPK2 intronic region; as this ARE sequence is conserved in humans (named intronic ARE: 5′-gTGACTCAGCg-3′), we analysed all three potential sites by studying NRF2 occupancy via ChIP-qPCR analyses. We used DLD1 cells to immunoprecipitate endogenous NRF2 and to interrogate its ability to bind to the three potential AREs. The analysis was performed by comparing the amount of material immunoprecipitated with anti-NRF2 antibodies in WT and NRF2-KO DLD1 cells (Figure 1d, left panel). Additionally, we compared the amount of material immunoprecipitated with anti-NRF2 or anti-IgG in DLD1 cells (Supplementary Figure S1I). Our results showed that at basal conditions NRF2 binds to the ARE1 and to the intronic ARE, but not to the more distal ARE2 sequence. Furthermore, we tested whether activation of NRF2 leads to an increase in its binding to the HIPK2 locus. To do so, we used NRF2-GOF DLD1 cells as a model for NRF2-sustained activation and we compared them with DLD1 WT cells, and found that NRF2 activation leads to an enrichment of NRF2 bound to the HIPK2 locus (Figure 1d, right panel). Finally, to address the potential functional relevance of the two identified sites, we used a luciferase-based genetic reporter assay. We cloned the proximal promoter of HIPK2 and the ARE-containing intronic region of HIPK2 upstream and downstream of the luciferase gene, respectively, and individually mutated the promoter ARE1 sequence and the intronic ARE sequence. We transfected these constructs into RL-34 cells (which are highly responsive to NRF2 inducers53) and tested their response to the NRF2 inducer TBE-3154, 55 (Figure 1e). We used plasmids containing either the promoter of NQO1 fused to luciferase (WT) or the promoter of NQO1 with a mutated ARE sequence fused to luciferase (MUT)56 as a positive and a negative control, respectively. Our results showed that while the ARE1 sequence situated within the HIPK2 promoter does not control luciferase expression, the ARE sequence within the intronic region of HIPK2 is responsible for the TBE-31-mediated induction of luciferase, highlighting the functional relevance of this ARE sequence.
Together, these results show that NRF2 regulates both basal and inducible levels of HIPK2 at the transcriptional level via an intronic ARE sequence. The identification of a functional intronic ARE, although rare, has been recently reported for another NRF2 target gene.57 To our knowledge, this is the first demonstration of a transcriptional mode of regulation for HIPK2.
HIPK2 supports NRF2 antioxidant response
Having demonstrated that HIPK2 is regulated by NRF2, we then studied whether HIPK2 affects NRF2-dependent responses. First, we found that HIPK2 overexpression promoted the accumulation of a nuclear, lambda phosphatase-sensitive form of NRF2 (in both endogenous and overexpressed NRF2) (Figure 2a; Supplementary Figure S2A). These results suggest that HIPK2 can activate NRF2 by promoting its nuclear accumulation, in a similar way as exposure to the oxidant H2O2 does (Supplementary Figure S2B). To test this possibility, we compared wild-type (WT) and HIPK2 knockout MEF cells and found that HIPK2-deficient cells had lower basal protein levels of NRF2 and the NRF2 target NQO1 and GSTM1 (Figure 2b), as well as an impaired induction of NQO1 after SFN treatment, as measured by enzyme activity (Figure 2c). Furthermore, HIPK2 reconstitution rescued (i) the basal mRNA levels of NRF2 target genes (Figure 2d) and their induction (in response to oxidants) (Supplementary Figure 2C) without affecting the mRNA levels of NRF2 itself, and (ii) the basal NRF2 protein levels, and its response to oxidants (measured by induction of NQO1 and HO1 levels upon exposure to H2O2) (Figure 2e). In these experiments, we used HIPK1/2-double knockout MEF cells to avoid potential compensation from HIPK1;2, 3, 58 similar results were obtained in single HIPK2 knockout cells (Supplementary Figure S2D). Interestingly, the effect of HIPK2 on NRF2 is kinase dependent, as a HIPK2 kinase-deficient mutant form (KD) did not rescue the basal NRF2 levels or the NRF2-mediated response (Figure 2e). However, based on our results we cannot distinguish between the effect of HIPK2 on NRF2 being direct or indirect.
To test the relevance of HIPK2 in the regulation of NRF2 in human cancer cells, we produced CRISPR-mediated HIPK2 knockout cells. We found that HIPK2 knockout decreased NRF2 levels in both H1299 and A549 lung cancer cells (Figure 2f). These results were confirmed in various cell lines using shRNAs against HIPK2 (Supplementary Figures S2E and S2F).
To test whether HIPK2 regulates NRF2 response in vivo, we analysed Hipk2, Nqo1 and Nrf2 mRNA levels from livers of wild-type and HIPK2 knockout mice treated with a single dose of the NRF2 inducer TBE-31. Compared to wild-type mice, HIPK2-deficient mice exhibited impaired induction of Nqo1 by TBE-31 (Figure 2g), without significantly affecting Nrf2 mRNA levels (Supplementary Figure S2G).
These results establish that HIPK2 contributes substantially to the NRF2-mediated responses both in cells and in vivo. Moreover, they highlight the potential role HIPK2 may play in ensuring a robust adaptive response against oxidative stress and xenobiotics. Of note, this new link between HIPK2 and oxidative stress responses is conserved throughout evolution, as a recent study demonstrated that in C aenorhabditis elegans, HPK-1 (the single homologue of HIPKs), confers resistance to oxidative stress.59
Physiological relevance of the crosstalk between HIPK2 and NRF2
NRF2 is well-characterised as being cytoprotective in healthy tissue. In clear contrast, NRF2 increases resistance against a wide variety of chemotherapeutic drugs in malignant tissue. The fact that HIPK2 positively regulates NRF2 suggests that this HIPK2/NRF2 axis could represent a new pathway by which HIPK2 prevents tumour initiation. However, the existence of such axis provides a means by which HIPK2 might, via NRF2, play a hitherto unrecognised role in mediating survival of malignant cells upon challenge with chemotherapeutic drugs.
To address whether HIPK2 affects cell responses to chemotherapeutic drugs via NRF2, we used two different approaches. First, we reconstituted HIPK1/2-KO cells with HIPK2 (or with an empty vector) and exposed both isogenic cell lines to increasing concentrations of the commonly used chemotherapeutic drug doxorubicin. We found that, compared to HIPK1/2-KO MEFs, cells reconstituted with HIPK2 exhibited higher cell viability (Figure 3a) and a striking reduction of apoptosis (measured by PARP cleavage) in response to doxorubicin, correlating with higher levels of NRF2 and NQO1 (Figure 3b). In full agreement with the apoptosis data, HIPK2-reconstituted cells were more resistant to doxorubicin (measured by cell viability), and this resistance was significantly reduced by NRF2 knockdown (Figure 3c). Second, we knockedout HIPK2, NRF2 or both genes in H1299 lung cancer cells, and measured their sensitivity to cisplatin compared with wild-type cells. HIPK2 knockout (or knockdown) increased the sensitivity to cisplatin as seen by a colony-formation assay (Figure 3d). Furthermore, whereas knockout of NRF2 sensitised cells to cisplatin, a double knockout of both NRF2 and HIPK2 did not increase drug sensitivity further as shown by a cell viability assay (Figure 3e).
These results confirm that HIPK2 can promote cell survival upon challenge with chemotherapeutic drugs; this, together with the involvement of the well-established pro-survival factor NRF2 makes a strong case for the idea that, by activating NRF2, HIPK2 could support cancer cell survival.
Conclusions
Herein, we demonstrate for the first time that HIPK2 is regulated at the transcriptional level and that HIPK2 is an NRF2 target gene. This is a notable finding because most HIPK2 regulatory mechanisms described to date rely on posttranslational modifications and thus, our results add an extra regulatory layer to the control of HIPK2 activity. Additionally, our data place HIPK2 as a critical kinase, shaping the NRF2 response. This is important in the cancer biology field for two reasons: First, our discovery reveals HIPK2 to be a common apical regulator of two major stress regulated pathways, NRF2 and p53, and thus a decisive factor controlling cancer cell fate by being coupled to both cell death and cell survival. It is important to highlight that although HIPK2 can protect healthy tissue against tumour initiation by promoting both DNA repair17, 22 and cytoprotection (shown in this study), activation of the same pro-survival pathways in malignant tissue could lead to aberrant cell survival and enhanced chemoresistance (see model in Figure 4), particularly when apoptotic pathways are impaired (that is, in the absence of a functional p53). In this context, it will be important in the future to address whether HIPK2 plays opposing roles depending on the stages of tumour development (for example, preventing initiation but accelerating progression) as has also been proposed for NRF2.60 Interestingly, HIPK2 also controls the levels of Notch1,61 a well-known factor with a dual role in cancer, which in common with NRF2, can act as both tumour suppressor and oncogene, depending on the context,62, 63, 64, 65 adding strength to the idea of a context-dependent role for HIPK2 in cancer. Second, our data suggest that inhibition of HIPK2 could be a plausible mechanism by which the NRF2 pathway could be suppressed, thereby providing a new strategy to overcome NRF2-associated resistance to therapies in malignant cells. It is recognised that aberrant-sustained activation of NRF2 can promote chemoresistance and radioresistance, and therefore, inhibition of NRF2 in these settings should increase the efficacy of anticancer therapies.
In summary, our results provide new information supporting the already well-established tumour suppressor role of HIPK2, and also could explain how under certain conditions (for example, cancer cell chemoresistance due to upregulated NRF2) HIPK2 might provide cancer cells with a survival advantage.
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Acknowledgements
We thank Prof Donna D Zhang (University of Arizona) for providing plasmids; Prof Issay Kitabayashi (National Cancer Center Research Institute Tokyo) and Prof Masayuki Yamamoto (Tohoku University) for cell lines; Prof Shyam Biswal (Johns Hopkins University) for sharing their ChIP-seq data; Prof Stephen M Keyse (University of Dundee) for critical reading and insightful comments on the manuscript. We are extremely grateful to the Medical Research Institute of the University of Dundee, Cancer Research UK (C52419/A22869), the Ninewells Cancer Campaign, Tenovus Scotland (grant number T14/62), Reata Pharmaceuticals and Stony Brook Foundation for financial support.
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Torrente, L., Sanchez, C., Moreno, R. et al. Crosstalk between NRF2 and HIPK2 shapes cytoprotective responses. Oncogene 36, 6204–6212 (2017). https://doi.org/10.1038/onc.2017.221
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DOI: https://doi.org/10.1038/onc.2017.221
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