P53 and Glycolysis in SCCHN

Discussion

Our unit recently published a study on the role of p53 in regulating energy metabolism in SCCHN cells and it has been observed that, loss of p53 function whether through mutation or RNAi-mediated downregulation, displayed a lack of flexibility, and the cells becoming more dependent on glycolysis (112). In this context, we are interested to find out, whether any other proteins, directly or indirectly activated by p53, involved in maintaining the high glycolytic status in SCCHN. Two proteins came to our attention; TIGAR and HK-2, both play a crucial role in glucose metabolism, the first one limits glycolysis and the second one initiates glycolysis by converting glucose into 6GP. Having known about the metabolic roles of each of these proteins, still there are few unanswered questions about the individual TIGAR and HK-2 expression patterns and any possible associations exist among the three proteins in SCCHN cancer cells. For those seeking support with similar research, biomedical science dissertation help can provide valuable insights into these complex interactions.

With the above questions in our mind, our primary aim of this thesis was to analyse the expression of p53, TIGAR, and HK-2 proteins in our selected samples of SCCHN. The secondary aim was to examine whether any association exist between the expression of these proteins and various clinical variables such as anatomical sites, T stage, N stage, differentiation, nerve invasion, vascular invasion, extracapsular spread, and also patinet outcomes (survival). Also, the expression levels of these proteins were analysed to see whether they influence each other's expression.

TIGAR (TP53 induced Glycolysis and Apoptosis Regulator) was identified as a novel p53 target gene in 2005 (195). To the best of our knowledge, this is the first study investigating TIGAR expression in SCCHN. In our cell lines studies, ( described in section 3.1), we observed highest levels of TIGAR expression are associated with wild type p53, whether expressed from a lentiviral vector in a p53 null line, or comparing RNAi-mediated knock-down in a p53 wild-type line, or even comparing two essentially isogenic lines from a single patient obtained at different times, with one harbouring wild type p53 and one possesing a p53 mutation (UM-SCC-11A and 11B respectively).

Our results of higher TIGAR expression in the cell lines with wild-type p53 compared to the mutant p53 cell lines are in accordance with the notion of p53-dependent TIGAR expression in our samples. As the name suggests, TIGAR (a p53-inducible regulator of glycolysis and apoptosis) is a p53-dependent target gene, and we have observed the connection between TIGAR expression and retention of wild type p53is potentially retained in our SCCHN cells.

High TIGAR expression in our p53 null cell lines could be explained on the basis that TIGAR expression is frequently uncoupled from the activity of wild-type p53 (282) and higher TIGAR expression in cancer cell lines is possible, which is not driven by p53, but occurs in a p53 independent mechanism via the action HIF1α and HK-2 (discussed in section 1.6.3.2). Thus, TIGAR has two functions: p53 dependent Fru-2,6-Bpase activity that is manifest in normoxic, hypoxic, and glucose-limited cells and functions to promote PPP, generate NADPH, and limit ROS, and p53-independent hypoxia-induced activity that depends on HIF1α and glucose, and further involves mitochondrial localisation, binding, and activation of HK-2 (205).

In our immunohistochemical analysis, TIGAR protein detected expression in 73% of samples in TMA set 1, whereas in TMA set 2, we observed TIGAR expression in all of the samples. The role of TIGAR in tumour survival is based on its ability to inhibit both apoptosis and autophagy (215). It has been observed that, TIGAR expression reduced ROS levels and protected from ROS-sensitive apoptotic responses, such as those induced by p53(196). Increased expression of TIGAR has been detected in many cancer types, consistent with a role of antioxidants in tumour progression (199), and TIGAR has been reported to mediate human cancer aggressiveness, although the mechanism is unclear (283).

Accumulation of ROS is one of the main sources of oxidative stress within the cells, and it has the harmful effects of arresting cell cycle or leading to cell death (284). In mammalian cells, NADPH provides the major reducing power and protects the cells from the oxidative stress and rapidly proliferating cells thus requires NADPH for normal function, proliferation and survival (285). In line with the pro-survival role of NADPH, TIGAR overexpression was observed in nasopharyngeal carcinoma (NPC) cell lines, and it was linked to increasing cellular growth, NADPH production and invasiveness of NPC cell lines while TIGAR-knockdown either by inhibiting c-Met tyrosine kinase (285) or through NF-κB pathway (286), inhibited proliferation NPC cells (284).

It has also been observed that, TIGAR induces aggressive breast cancer by way of reciprocal metabolic changes with reduced glycolysis in tumour cells and increased glycolysis in stromal cells such as fibroblasts (283). A high immunohistochemical expression of TIGAR (74.3%) was observed in 113 cases of primary invasive breast cancer patients along with the expression of p53 (27.5%), synthesis of cytochrome c oxidase (SCO2) (84.1%), and COX (73.4%) (287). In the same study, it has been observed that, high p53 expression was significantly associated with the low expression levels of SCO2 (P=0.008) and TIGAR (P=0.007) and it has been suggested that, high p53 expression could promote aerobic glycolysis in breast cancer via modulation of mitochondrial enzymes (SCO2 and COX) and TIGAR (287). A similar immunohistochemical analysis of 110 cases of primary gastric cancer identified an inverse correlation between p53 and TIGAR and suggested that high p53 expression could be associated with the promotion of glycolysis in gastric cancer via the modulation of TIGAR expression (288). Both studies on breast cancer and primary gastric tumours showed that p53 expression was negatively correlated with TIGAR expression (287, 288) and analysis of our IHC data did not reveal any significant association between inferred p53 mutant staus and TIGAR expression in TMA set 1 (P=0.580) (section 3.2.3). Unfortunately, it has not been possible to address this in the TMA set 2 samples because of the oveall strong staining of TIGAR in all the slides.

TIGAR expression was investigated in twenty-two matched colorectal cancer patients, and the results identified the upregulation of TIGAR in colorectal patients with the additional finding of a noticeable increased TIGAR expression at the mRNA and protein levels in stage Ⅱ and stage Ⅲ colorectal cancer (289). Their immunohistochemical analysis revealed that, 68% of colorectal tumour tissues were strongly positive for TIGAR staining, however weak to moderate TIGAR staining was also noted in the nearby normal tissues (289). Another interesting finding in the above study is the nuclear localisation of TIGAR in their immunohistochemical staining, whereas the majority of the studies, including our study, showed cytoplasmic staining of TIGAR (section 2.4.4).

Contradicting the above studies which showed increased TIGAR expression correlates with increased cellular growth and invasiveness (284, 286, 289), a study on 79 patients with primary non-small cell lung cancer (NSCLC) which compared SUVmax (maximal standardised uptake value determined through PET imaging) with TIGAR identified that SUVmax was negatively correlated with the TIGAR expression and decreased expression of TIGAR was strongly correlated with a poor clinical outcome (290). TIGAR was observed to act on cell cycle arrest, and it was proposed that, TIGAR promoted p21-independent, p53-mediated G1-phase arrest in cancer cells (291). The consensus from the above studies can be interpreted as, even though TIGAR limits ROS-associated apoptosis and autophagy, the ability of TIGAR to promote cell survival is cell and context-dependent (205).

The other protein of interest in this study was p53 and our immunohistochemical analysis nferred that p53 was likely to be mutated in approximately 76.9% of samples from TMA set 1 and in 65% and 50% of samples from the tumour core and advancing front samples of the larynx and hypopharynx cohort of TMA set 2. This is similar to the estimate of 84% for p53 mutation obtained by TCGA network analyses of 279 SCCHN cases (40). In lymph nodes samples of larynx and hypopharynx, the inferred p53 mutant rate was 83.9%. Our inferred p53 mutation rate is also similar to another large series study (724 primary SCCHN patients), where they identified high p53 expression of 71.7% in their samples by doing immunohistochemical analysis (292).

Another important observation was the relationship between p53 expression in oropharyngeal samples. Although we would expect an inverse relationship between the presence of HPV DNA and the presence of TP53 mutations in oropharyngeal squamous cell carcinoma (293), in our limited number of oropharynx samples, inferred p53 mutation rate was 72.6% and 50.00% for tumour core and advancing front samples, respectively. This seems surprisingly high, since that we might expect p53 in these samples to frequently be wild-type as a result of HPV infection in many of this cohort, both positive and negative groups.

The expression of wild-type p53 in HPV related oropharyngeal SCC (OPSCC) (40), creating a possible confounder as HPV driven tumours commonly have a favourable prognosis (191). Regarding survival, HPV positive head and neck tumours differ from HPV negative tumours that they have improved survival, with a 60% lower risk of death (294). We found a significant survival difference between HPV +ve and -ve cases, where HPV +ve cases showed a better survival period as compared to HPV negative cases (P=0.001) (section 3.3.5.2). Considering the overall prognosis of HPV related to head and neck tumours, even though many studies have indicated a relatively better outcome for HPV-positive SCCHN patients, particularly those with tonsillar cancer (175, 295-297), the reports are not unanimous (298, 299).

Our justification of using IHC to find the inferred p53 mutation rate is based on the following facts; wild-type p53 in unstressed cells has a short half-life, and as a result, it is found at very low concentrations in all healthy cells, so wild-type p53 is almost undetectable in the routine immunohistochemical analysis (300). Compared to wild-type p53, the mutant p53 has a long half-life and tend to accumulate in the nucleus, so that it can be a stable target for immunohistochemical detection (301, 302). Point mutations often increase the half-life and stabilise the p53 protein, which further allows detection by immunohistochemical methods (300). Thus, the detection of p53 by using IHC in tumours is almost synonymous with the presence of a mutation, and immunohistochemical analysis of p53 expression is utilised as a surrogate marker for its mutation analysis (303-305). Not all mutations in the TP53 gene result in protein stabilisation. For example, mutations resulted due to gene deletion or truncation of the protein (nonsense and frameshift) do not cause protein accumulation (300). This is the reason we have considered the complete absence of p53 expression as an ‘inferred nonsense mutation’ and added to the mutant category. Based on this fact, our ‘inferred mutant’ category includes cells showing either >5% or complete absence of p53 expression. We decided to have the cut off value as >5% for the inferred mutant category, and we have noted similar cut off value (5%) in a study where they evaluated p53 response to short-term preoperative radiotherapy and patient survival (306). But, various authors have used a different cut off value for the percentage of p53 nuclear staining to determine p53 mutation (188, 190, 191, 292, 307). In a study to analyse fifty-five cases of oral squamous cell carcinomas, the authors increased their cutoff value for p53 immunoexpression to 25%, and they observed increased overexpression of p53 (64%) in their samples (308). They suggested that 25% of p53 immunopositivity appears to be a good cut off value to predict TP53 mutations (308).

The third biomarker used in our study was HK-2, and overall high HK-2 expression was identified in our western blot analysis. Our immunohistochemical analysis reported 42.4% and 28.3% of strong HK-2 expression in the tumour core and advancing front samples of larynx and hypopharynx, respectively. Our Oropharynx samples showed strong HK-2 expression of 54.8% in the tumour core and 38.7% in the advancing front samples. The lymph nodes displayed strong HK-2 expression of 61.3% in the larynx and hypopharynx groups and 79.3% in the oropharynx group.

HK-2 expression is rarely observed in normal tissues, and high levels of HK-2 expression has been reported in various solid tumours, including colorectal tumour (309), gastric cancer (310), hepatocellular carcinoma (311), ovarian cancer (312), and pancreatic cancer (313), indicating the important role of HK-2 in tumorigenesis (314).

In SCCHN, HK-2 is highly expressed, implying that SCCHNs are “glycolytic tumours” (315). Increased HK-2 expression was observed in oral squamous cell carcinoma along with cancer metabolism-related proteins like GLUT-1, lactate dehydrogenase A (LDHA), transketolase-like-1 (TKTL1), and mitochondrial enzymes (succinate dehydrogenase SDHA, SDHB, and ATP synthase) and insulin-like growth factor receptor (IGF-1R) (316). It was observed that, squamous cell carcinoma of the tongue with higher migratory /invasive capacity had increased levels of HK-2 expression and that HK-2 overexpression promoted the proliferation, migration, and invasion of tongue cancer cells, whereas HK-2 knockdown inhibited these processes both in vitro and in vivo (317). Overexpression of HK-2 was observed in laryngeal cancer, and it has been suggested that, high HK-2 expression might be related to the progression of laryngeal cancer (318).

The three proteins p53, TIGAR and HK-2 are interconnected to each other and it influence the expression of other proteins which can be explained by the following facts; As a p53-inducible protein, TIGAR regulates mitochondrial HK-2 localisation, and this TIGAR-HK-2 complex further upregulates HK-2 and HIF1-α activity resulting in reduced ROS production which protects the tumour cell death under hypoxic condition implying that p53 could be an important key regulator for HK-2 mediated tumorigenesis (205, 279, 315).

Our attempts to explore the inter-relations of these proteins were based on the following observations; the association between p53 and HK-2 expression was analysed in our larynx and hypopharynx samples, and it was observed that, there was a significant association between tumour core (P=0.003), and advancing front (P=0.047) samples. This observation suggested that patients with strong HK-2 are more likely to have inferred mutant p53 status in the larynx and hypopharynx samples. In 1997, Mathupala et al. first reported a possible association between mutant p53 and HK-2 expression in tumour cells, where they identified two functional p53 motifs within the HK-2 promoter and suggested that the mutated p53 interacts with the HK-2 promoter in cancer cells to activate transcription of HK-2 (280). With their experiments, it was proposed for the first time that mutant p53 was able to induce a gain-of-function and transactivate a gene (HK-2) that was essential for maintaining high glycolytic activity, and therefore the survival of a rapidly growing tumour (280).

Following the observation of an association between p53 and HK-2, TIGAR expression was correlated with p53 expression in TMA set 1, and there was no significant association between p53 and TIGAR expression (P=0.580). Because of the overall strong staining of TIGAR in TMA 2, we were not able to identify any correlation between TIGAR and the other two proteins p53 and HK-2.

In our second part of the IHC analysis, the expression of TIGAR, p53 and HK-2 was correlated with the known clinical variables to see whether any significant association exist between the individual protein expression and any one of the variables. We observed a significant association between p53 expression and anatomical sites (P=0.011 and P=0.015) in oropharynx samples (both tumour core and advancing front). There is good evidence of differing biological behaviour of SCCHN and response to treatment depending on the primary anatomical site (319). But, a systematic review and a meta-analysis failed to provide conclusive evidence about the prognostic value of p53 expression in patients with SCCHN arising from larynx, oropharynx, hypopharynx, or oral cavity (181).

There was a significant association between p53 expression in tumour core samples of the oropharynx and the event (death) status (P=0.017). Though there is good evidence from the literature suggesting accumulation of p53 that can serve a prognostic role of overall and disease-specific mortality in SCCHN (320), our study did not reveal any significant difference in the survival rate between the wild and mutant p53 expression both in TMA set 1 and 2.

We also observed a significant association between HK-2 expression in tumour core samples of larynx and hypopharynx and anatomical sites (P=0.017). There was an association between HK-2 expression in tumour core samples of larynx and hypopharynx with N stage (P=0.023), differentiation (P=0.041), and vascular invasion (P=0.034). There was an association between HK-2 expression in lymph nodes samples of larynx and hypopharynx with nerve invasion (P=0.020). In our study, HK-2 expression was slightly higher in the tumour core samples (42.4%) than in the advancing front (28.3) of larynx and hypopharynx group. In a breast carcinoma study (321), HK-2 immunoreactivity was noticeably present in the advancing front of the tumour which is generally considered the most biologically active part of the carcinoma and is most likely to decide the outcome of the disease (322).

Targeting altered metabolic pathways related to carbohydrate metabolism is one of the promising anti-cancer strategies (323). There is emerging evidence that targeting TIGAR and its downregulation favours antigrowth properties (285, 324-326). Resveratrol (3,5,4’-trihydroxystilbene), a phytoalexin, has been identified to exhibit its anti-cancer effects through a variety of mechanisms, and one among them is downregulation of TIGAR (327). An RNA-directed nucleoside analogue, ECyd is known to possess the anti-cancer activity, and it was observed that, ECyd also could induce significant downregulation of TIGAR (328). There is convincing evidence that aerobic glycolysis constitutes the metabolic signature of SCCHN, and this metabolic switch is driven by the mutational loss of wild-type p53 function (329). Thus, p53 becomes the promising target, which either restore wild-type p53 activity or inhibiting mutant p53 oncogenic activity could provide a potent strategy to treat malignant diseases (330).

The approaches to activate endogenous wild-type p53 include the use of gene therapy to introduce wild-type p53 or modified adenovirus to kill tumour cells with mutant p53, the use of chemoradiation to activate endogenous wild-type p53, and the use of synthetic peptides or nongenotoxic small molecules to activate wild-type p53 (331). Nutlins are the potent and selective low molecular weight inhibitors of MDM2-p53 binding which activate the p53 pathway and suppress tumour growth in vitro and in vivo (332). Another small molecule which reactivates wild-type p53 is RITA (Reactivation of p53 and induction of tumour cell apoptosis) could suppress tumour cell growth both in vitro and in vivo by inducing massive apoptosis in a p53-dependent manner (333). Restoring p53 function in those tumours expressing mutant p53 is even more challenging, and small molecules that refold some mutant p53 proteins and thus reactivate their wild-type functions that have been described (334).

Metabolic enzymes could be an attractive candidate for cancer therapy, but there must be a significant difference in the requirement for a given enzyme’s activity between cancer cells and normal proliferating cells, potential examples include GLUT1, HK-2 and LDH-A (335). Hexokinases, which catalyse the first committed step of glucose metabolism, are one of the promising target for anti-cancer therapy as many cancer cells overexpress HK-2, and preclinical studies demonstrated that the inhibition of HK-2 could be an effective cancer therapy (336). As most healthy adult cells do not express HK-2, its systemic ablation could selectively target tumour cells, and there is good evidence that HK-2 act on tumour cells without adverse physiological consequences (116). However, it is challenging to develop small-molecule inhibitors, which preferentially inhibit HK-2, as there are structural similarities between HK-1 and HK-2 (116, 217, 224, 229). The allosteric inhibition of HK-1 and HK-2 by their own product, G6P, and it could be utilised to target HK-2 (116). G6P could also be utilised to preferentially target HK-2, although G6P inhibits both HK-1 and HK-2, its inhibitory effect on HK-2 increases in the presence of orthophosphate, whereas HK-1 inhibition is reduced (217). Another promising agent for targeting HK-2 is 3-bromopyruvate (3-BP), whose biological function is based on the alkylation of free thiol groups on the cysteine residues of proteins (337). The small alkylating molecule, 3-BP, was discovered as a noval anti-cancer agent in vitro in the year 2000 and was published in 2001 (338). The inhibitory effects of 3-BP on the glycolytic pathway have been demonstrated in various in-vitro studies, which are mediated by covalent modification of HK-2 (339). 3-BP targets cancer cell’s energy metabolism, i.e., both glycolysis and mitochondrial oxidative phosphorylation to inhibit total energy (ATP) generation and depletes all energy reserves (337, 338). The action of 3-BP happens rapidly (within minutes) and with little or no effect on most normal cells or the animals as compared to the frequently used chemotherapeutic drugs, which may take a longer duration of time (weeks/months) to exhibit any significant changes (337). Therefore, 3-BP, as a pyruvate mimetic, is a potent, rapid and quite specific anti-cancer agent (219, 340). 3-BP was tested in pre-clinical models, where it was shown to be effective, eradicating advanced tumours in 19/19 animals, without harming the animals and without returning of the cancer during their life time (341). It has been reported that 3-BP is non-toxic to all sorts of vertebrates and certain failure cases were still reported in clinical trial (342). Apart from 3-BP, other agents that can inhibit hexokinases are 2DG (2-deoxy-D-glucose) (343), and Lonidamine (344).

The main limitations of our study were different cohorts of patient samples, choosing IHC for evaluating p53 expression, and practical difficulties in conducting metabolic studies. Due to the delay in construction of new head and neck TMA, we started the research by using readily available TMA which had samples of oral cavity and oropharynx (TMA set 1). The new TMA had different patient’s samples from larynx, hypopharynx, and oropharynx with different sets of clinical data (TMA 2), which made us evaluate the two TMAs separately. Selecting immunohistochemistry to investigate p53 was based on the fact that, most mutations change the conformation of p53, indirectly leading to a more stable protein which can then accumulate in tumour nuclei and subsequently can be detected by immunohistochemically (277). However, there are many problems in IHC that can result in false-negative or false-positive outcomes (303, 305, 345).

False-positive results can be due to stabilisation of wild type p53 by physiological stimuli such as hypoxia, oncogenic stresses, or DNA damage resulting from the free radicals released from the tumour-associated macrophages or following therapy, leading to positive staining in the absence of mutation (346, 347). On the other hand, false-negative results are possible because of truncating alterations, including nonsense, frameshift and splice site mutations, which result in lack of immunolabelling due to the absence of gene product (348). Erroneous splicing produces a truncated protein, which will have a shorter half-life and escapes IHC detection (349).

The common antibodies used (DO7, DO1, and Pab 1801) for p53 detection in IHC are unable to differentiate between mutant and wild-type p53 proteins (181, 350). Presently, there is no consensus for the most appropriate antibody for evaluating mutation associated p53 expression (351).

Another issue is the selection of cutoff point to dichotomize p53 positivity versus p53 negativity which varies from one positively stained cell to 50% positivity, and both extremes are difficult to justify from a biological perspective (181). The frequent occurrence of heterogenous upregulation of mutant p53 has been observed (352), and thus fixing a cutoff of 50% might exclude many mutant proteins that display low levels of p53 positivity (181). In future studies, it is essential to improve the reliability of p53 IHC as a surrogate method and one simple strategy is to combine p53 detection along with monitoring of p53 target gene expression (MDM2 and/or p21) (277). When cells expressing p53 but not expressing MDM2 and/or p21, regardless of the relative levels, likely to have mutant p53, whereas coexpression of p53 and MDM2 would be likely to have wild-type p53 proteins (181, 277).

Targeting cancer cell metabolism seems appealing at first glance because enzymes are attractive molecular targets; however, there are some significant issues when targeting glucose metabolism for anticancer therapy (335). Apart from the tumour cells, immune and stem cells can also perform aerobic glycolysis, and it can be hard for anti-metabolic therapies to differentiate between tumour and non-tumour cells (335). Most of the metabolic reprogramming studies were performed in cancer cell lines rather than intact tumours, and it is challenging to model an accurate tumour microenvironment in culture (111). Another challenge could arise from the metabolic plasticity displayed by cancer cells as there is a possibility that cancer cells could develop resistance to inhibition of a particular pathway through the expression of alternative isoforms or up-regulation of alternate pathways, such as gluconeogenesis (335). Despite these challenges and several unanswered questions in the field of cancer metabolism, our understanding of cancer metabolism has advanced noticeably in the recent years and is being used for the development of novel targeted therapeutic strategy (353).

Our unit has recently published a study, where they confirmed the role of p53 in the metabolic regulation in SCCHN cells and suggested that when cells display loss of p53 function, they can be sensitised to ionizing radiation by pre-treatment with a glycolytic inhibitor (112). Future experiments in a pre-clinical model are required to provide further evidence for the feasibility of therapeutic strategy.

We were recently awarded ODA Research Seed fund of £10,000 from University of Liverpool (2020/21 round) to conduct an animal study, collaborating with Adyar Cancer Institute, Chennai, and Sri Ramachandra Institute of Higher Education, Chennai, India. We propose to develop a preclinical model for oral cancer, and as a first step, we will test the tumorigenicity of our selected SCCHN cell lines in nude mice. With the collaboration between the University of Liverpool and Cancer Insitute and SRIHER, we hope to develop local expertise at Indian Institutions at growing and manipulating SCCHN cell lines as well as generating mouse models of cancer.

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In summary, this is the first study of TIGAR expression in SCCHN and our data suggest that, unlike some other cancers, the link between p53 and TIGAR expression is retained, at least in cell lines. HK-2 is expressed in all cell lines and the steady state levels do vary much between cells with different p53 status. It is therefore perhaps surprising that, we have detected some variability in HK-2 expression in tumour samples by IHC. Moreover, we have identified a strong association between HK-2 expression and p53-inferred mutant status in larynx and hypopharynx samples which suggests that loss of p53 function, which would be expected to increase glycolysis, and it is associated with increased expression of a key glycolytic enzyme. This is not suprising, but it is the first time that this asscoaition has been identified in SCCHN. Importantly, this may indicate that, targeting HK-2, for example with an inhibitor could prove particularly effective in p53 mutant tumours. These studies include the first attempt to characterise TIGAR expression in SCCHN, and it is combined with anaysis of HK-2 and using IHC to infer p53 status has revealed some intriguing associations. Ultimately, it is clear that, cancer cell metabolism is a highly attractive target for therapy. More promising results have been observed in anti-HK-2 treatments, especially with 3-BP. By creating a pre-clinical model, we are hoping to achieve the successful bench-to-bedside translation of basic scientific findings to novel anti-cancer interventions.