DOI: 10.31038/CST.2020524

Review Article

Stress of Working in Oncology

Oncology is a medicine area of high psychic investment. Working with cancer patients is a source of human and professional satisfaction but can involve high emotional costs [1,2]. High levels of burnout and compassion fatigue are reported by about 32% of oncologists [3,4] this percentage rises to 70% among people under 40 years [5]. High levels are also found among nursing staff with marked levels of emotional exhaustion [6]. Repeated exposure to suffering and loss, to the side effects and/or the failure of treatments to the end of life stages, to feeling overwhelmed by work, are among the causes of chronic distress that medical staff accumulate in clinical practice. Care of cancer can result  in emotional distress and exhaustion, loss of empathy, and demotivation from work [7]. Of no less importance is the “difficult” communications that are estimated at around 20,000 in the career of an oncologist [8].

Prevalence of COVID-19 and Risk of Infection

It is well known in the history of infectious diseases that health workers are those who pay a high cost. On the one hand testifies to the tendency of healthcare personnel to take risks for patient care, on the other highlights how often they are confronted with epidemics without adequate safety systems [9]. COVID-19 has been the most catastrophic pandemic after the 1918 H1N1, known as the “Spanish flu”. The Italian data of April 23, 2020, by the Istituto Superiore di Sanità (ISS) [10], show that 11% of the symptomatic infected people are health workers, with a median age of 48 (vs 62 years, total case median age), 69% women, with a lethality rate of 0.4%. The increased exposure of health personnel is also confirmed by the data of the Italian National Institute for Accident Insurance at Work  (INAIL)  [10]. The Health and Social Care sector (hospitals, nursing and rest homes, etc.) reports 72.8% of cases. Of the 28,381 SARS-CoV-2 occupational infections reported between the end of February and April 21, 2020, 71.1% are women. The median age of 48, 45.7% are health technicians (nurses, physiotherapists), 18.9% socio-health workers, 14.2% doctors, 6.2% social workers, 4.6% unqualified staff related to health services and education. The territorial analysis shows a distribution of complaints of 52.8% in the North-West (Lombardy 35.1%), 26.0% in the North-East (Emilia Romagna 10.1%), 12.7% in the Centre (Tuscany 5.5%), 6.0% in the South (Puglia 2.6%), 2.5% in the Islands (Sardinia 1.3%). The monitoring also detects 98 reports of accidents with a fatal outcome following COVID-19 (about 40% of the total deaths at work reported to INAIL in the period under review), median age 59 years, 79.6% are men. The territorial analysis shows a distribution with 54.1% of deaths in the North-West (Lombardy 36.7%), 13.3% in the North-East (Emilia Romagna 9.2%), 10.2% in the Centre (Marche 4.1%), 20.4% in the South (Campania 9.2%) and 2.0% in the Islands (Sicily 2.0%). The category of health technicians (nurses, physiotherapists) is the most affected (15%), followed by doctors, social and health workers, and social workers (all three professional categories, 13%).

The data on the place of exposure of the contagion (sample n = 5000) in the period 1-23 April 2020, show as most at risk the Healthcare Residences (RSAs), then the Communities for Invalids and Retirement Homes (44.1%), family (24.7%), hospitals and clinics (10.8%), workplace (4.2%) [11]. The Italian data are consistent with the Chinese and North American ones which identify a category at risk of contracting COVID-19 in healthcare personnel, with percentages of 3.8% of the total cases in China and 19.0% in the USA. A higher prevalence of women, younger individuals, and a lower lethality, probably due to the higher number of swabs carried out, are the characteristics that differentiate health workers from the total sample of subjects who contracted the virus [12,13].

Challenges Posed by COVID-19 to Healthcare Workers in Oncology in the Phase of Strong Epidemic Spread

Our country and the National Health System (NHS) were not prepared for this pandemic unlike others like China and South Korea who had faced other flu epidemics such as SARS and MERS. Italy had never found itself having to implement such an impressive health response plan. In phase I, while many hospitals were transformed into intervention hospitals or HUBs for COVID-19, the staff who worked with cancer patients faced professional  and  personal  problems.  The scarcity of adequate Personal Protective Equipment (PPE), the uncertainty regarding the mode of transmission  of  the  virus  and the related protective behaviours, the consequences of the delay in recognizing some positive  patients,  the  responsibility  of  patients at risk for age and immune conditions, the first contagions among colleagues with consequent worry and workload, the fear of infecting their family members, the lack of swabs and reagents constituted the initial scenario. In an “unexplored terrain” where little was known about the virus and even less about possible therapies, the oncological health workers participated together with the others in the trauma and mourning, powerless witnesses of the surge of infections, the wave of deaths, the collapse of the Intensive care, patient transfers to other contexts.

Working in oncology they should have been used to considering death in the background, but this was “another” disease. The high rate of contagion among health professionals [14], the median 10 days from diagnosis to death, the lethality rate reported as preliminary data by the National Institute of Statistics-ISTAT [15] showed an increase in deaths equal to or greater than 20% compared to the average figure for the same period in the years 2015-2019, which made COVID-19 a different phenomenon. Health workers were personally touched by the fear of contagion, by the death of colleagues, patients, and loved ones, feeling exposed, mortal, and fragile [16,17]. “Am I part of the cure or am I part of the disease?” a doctor asks himself, stressing the conflict between a sense of responsibility for one’s job and that of one’s family [18]. The fear of “taking the virus home” is not unfounded if we think that 41% of COVID-19 cases in Wuhan resulted from hospital- related transmission [19] and that Italian data show how the exposure to the virus occurred in 24.7% of cases in the family environment. [10] A fear that can trigger primitive reactions of social stigma in an insidious way. This explains the paradox described in other epidemics whereby what are called “heroes” for the value attributed to their work [20] may be feared by some as potential “greasers” capable of bringing the virus into their condominiums or homes [21].

Due to the risk of moral  injury,  that  particular  phenomenon that occurs when you know that “you should do something but you cannot do it” with a consequent sense of guilt, betrayal, and anger [22] is likely that oncological health workers have not been as exposed as the colleagues who worked in the front line or in the triage forced  for lack of oxygen, fans or beds to give priority to some categories  of patients and not to others. However, more nuanced feelings of “moral disorientation” have emerged related to both the fact that “the questions of Medical Oncology for  the  correct  approach  in the management of COVID-19 are multiple and unresolved”, [23] and to the “transgression”  of evidence-based reference paradigms,  in the absence of consolidated scientific evidence on the effects of COVID-19 on patients and cancer therapies, and the “distraction effect “consequent to the NHS focus on the pandemic [24-26]. In any case, the oncologist had to face the daily task of “balancing the value of cancer treatment with competing risks during a time of declining resources” with ethical and logistical challenges to clinical standards and humanism [23].

Impact of COVID-19 on the Mental Health of Operators and Family Members

We do not yet have prevalence data on peritraumatic distress from COVID in cancer healthcare professionals although data on a general population sample show that a third of people experienced symptoms of mild/moderate and severe peritraumatic distress [27].

That health workers can report mental health problems, personal and professional emotional pressure during epidemics and pandemics, even to a greater extent than that found in the general population, which is a phenomenon described in the literature on SARS, MERS and H1N1 and confirmed by recent studies on COVID-19. The prevalence of psychological and psychopathological disorders can vary according to the pandemic phase, with higher peaks in the initial periods [28] when the initial impact can cause a higher distress response. A single-center study on 5062 COVID hospital workers in Wuhan highlights distress (29.8%), depression (13.5%), and anxiety disorders (24.1%). The variables associated with psychic suffering were gender, years of seniority, the presence of chronic illness, and the history of mental disorders, having positive or suspicious family members, and evaluating the Hospital Institution as a tutelary and supportive [29]. Another study of 1257 health worker (60.8%, nurses and 39.2% doctors) in hospitals in China (60.5% in Wuhan and 39.5% outside Wuhan), of which 41.5% front line operators, reported depressive symptoms (50.4%), anxiety (44.6%), insomnia (34.0%) and distress (71.5%). Being nurses, women, working on the front line, in Wuhan Hospitals was associated with higher levels in all measured mental health dimensions. Healthcare professionals directly involved in the diagnosis and treatment of COVID-19 patients have a high risk of depressive symptoms, anxiety, insomnia, and distress [30].

An Italian study carried out between 27 and 31 March 2020 on 1379 health workers from 20 Italian regions, using an avalanche sampling technique, highlights symptoms of post-traumatic stress (49.4%), symptoms of severe depression (24.7%), anxiety (19.8%), insomnia (8.3%) and high distress (21.9%). Female gender and young age are risk factors for all study outcomes; having been personally exposed to the infection is a risk factor for depression, working in the front line is positively related to symptoms of post-traumatic stress, having a dead colleague, hospitalized or in quarantine was significantly associated with high levels of insomnia, depression and perceived stress, finally being nurses or social and health workers is a risk factor for severe insomnia [31].

Since being in hospital could favor the possibility of being infected it is legitimate to hypothesize the concern of family members considering the risks involved in the work of their relatives. Little has been studied in the psychological suffering of family members of health workers in epidemics. A recent survey [32] carried out from 10 to 20 February 2020 on 822 family members of health workers who worked in 5 hospitals in Ningbo, China, showed high levels of distress with  a prevalence of anxiety disorders (33.7%) and depressive symptoms (29.4 %). Significant differences were associated with characteristics of the health care professional’s work such as availability of adequate PPE, the number of hours worked, contacts with positive or suspect patients and personal characteristics of the family member, such as the number of hours focused on COVID. Parents and children, compared to their spouses, were at greater risk of distress. These data require attention for correct allocation of resources for dedicated interventions, as required by the Guidelines for interventions on the psychological crisis for the pneumonia epidemic due to new coronavirus infection. The Chinese National Health Commission treats family members of healthcare professionals as the 3rd priority group.

What Resources and What Interventions?

A first consideration regards the timeliness with which researchers from around the world reacted to the challenges posed by an unknown virus, the cause of a pandemic that has positioned itself in a few months among the main causes of death in different countries [33]. This reaction united the scientific community and witnessed the production and sharing of a large number of articles in peer-reviewed journals and sources such as bioRxiv and medRxiv, with a speed of publication unthinkable in normal times. Over 40,000 searches have been collected under the aegis of COVID-19 Open research Dataset (CORD-19) and uploaded to a site of the Allen Institute for Artificial Intelligence to allow data scientists and artificial intelligence experts to consult them [34]. Healthcare institutions in the USA [35] have set up a register of cancer patients for faster and wider sharing of data, as well as research groups and scientific associations from the oncology area in Italy.

All this has made it possible to identify new research paths and a better understanding of the phenomena in their evolution. And above all, it offers the knowledge bases to political and health authorities to develop and implement support actions for a category widely exposed to distress such as that of health workers. Peritraumatic distress is known to be associated with an increased likelihood of developing Post Traumatic Stress Disorder (DPTS) and other psychological symptoms in the years following natural disasters. A recent study highlighted that “to have received psychological support” was a variable associated with lower distress scores [27]. A first answer, at the level of available resources, is the psychoeducational material disclosed in the form of videos or informative material, produced by national and international Organizations and Agencies (e.g. CSTS, ISS, National Comprehensive Cancer Network – NCCN), which aims to support the well-being of healthcare professionals during the coronavirus epidemic or other epidemics [36,37].

In distress prevention programs one must not forget the importance of the role of the team leader and the type of leadership, of how they will be able to guide their collaborators in moments of uncertainty towards necessary changes, support them in the needs of reassurance, recognize their feelings of anxiety and condolence and encourage them in the development of group cohesion [38]. Cohesion is a powerful factor in the dynamics of a team, described as that particular “climate” that is perceived by observing a group at work, that “team spirit” characterized by a propensity for mutual support, even hard confrontation between members but always on a constructive and loyal group membership background. The “team spirit” is not easy to build from nothing in times of crisis and strong work pressure, therefore it would be desirable that the institutions in times of stability provided for the formation of leaders capable of proactively promoting a community of colleagues based on the value of the mutual support, particularly in critical areas such as oncology [39]. Other resources concern the activation by the health structures of remote psychological interventions dedicated to operators in the oncological area such as telephone helplines. The helplines operate with the first level of intervention which consists of accepting the motivation of the call and understanding its meaning. As a rule, a psychological triage follows to collect information in a short time on the nature and severity of the problem presented, on the resources available to deal with it. In some cases, a single contact that takes on counseling characteristics or a brief intervention on the crisis may be sufficient. However, in the presence of persistent symptoms, marked family difficulties, in work or in social life, with the risk of complications and/or suicide and/or evidence of major psychopathological disorders, a structured second- level intervention is carried out with a referral to a psychotherapist or psychiatrist [40,41].

Further resources for healthcare professionals  are  represented by the defusing and debriefing interventions developed in the field  of emergency psychology, are used in psycho-oncology in highly stressful critical situations that suddenly impact on the usual routine of the work team, imply the perception of a threat on a physical and/or emotional level, upset the feeling of control and interfere with the usual coping skills of individuals. Defusing can be defined as an immediate psychological first aid technique, of “sharing and emotional reworking related to a traumatic event” [42]. The Debriefing intervention for Stress from Critical Accidents is a more structured intervention to be carried out in the 48/76 hours following a critical event, in any case no later than two months from it. If indicated, it can also be offered remotely [43].

Communication and Relationship Challenges in the Care of Cancer Patients

In phase I, with a strong epidemic spread, the members of the cancer team found themselves managing new relationship situations with patients and their families, following the abrupt reorganization of care. Clinical experience and scientific publications have proposed new topics that may constitute communication challenges: the management of patients’ feelings of anxiety, abandonment or ambivalence towards the Hospital, which is seen as a safe base but also as a reservoir of infection; the communication of positivity for COVID-19 to a metastatic patient hospitalized for cancer treatment or to a patient who has recently received a cancer diagnosis; the communication to the relatives of the transfer to the COVID-19 ward or of intensive care of their relative; being intermediaries of highly emotional communications, telephone or video calls at the patient’s bed; answering difficult questions and finally adapting to new ways of telematic assistance.

During a pandemic emergency, personal protective equipment and social distancing can modify, limit, and alter the use of non- verbal communication and require an “enhancement” of the role of verbal communication in the patient’s medical relationship to convey truthful information and reassurance on the reasons of the multiple changes in procedures, giving support in critical phases of the care path and in the absence of support from family members, answering difficult questions and, last but not least, of importance for having conversations on end-of-life issues (such as the discussion on the Advance Care Planning and Decisions about Do-Not-Resuscitate Order also in a proactive version adapted to COVID-19). The importance was stressed in addressing Advance Care Planning during COVID-19 and of prioritize discussions about goals of care at the onset of serious acute illness [44]. Not a simple task in a country like Italy where half of the patients with metastatic cancer are unaware of the prognosis [45] and where the Early Treatment Provisions (DAT) are rarely discussed in clinical practice [46].

It is known how communication skills can be perfected through Evidence-Based training and which are associated with less burnout in the care staff, with greater satisfaction with the care received and positively with other outcome measures in patients [47,48]. The psycho-oncologist can provide, in an emergency, healthcare professionals with targeted counseling on the psychological aspects of critical communication steps. Health workers with training in communication skills are facilitated and adapt the skills acquired to new communication challenges. Untrained operators  can benefit from toolkits with behavioral indication  of what   it would be better to say or do in difficult relationship situations of COVID-19 [49]. Taking care of  communication and  relationships  is not secondary for health workers because, when everything is over, the perception of having been important reference and support figures for other bewildered and frightened human beings will have a comforting and protective effect on one’s own and their mental health [50].

Abbreviations

COVID-19: Coronavirus Disease 2019

CSTS: Center for the Study of Traumatic Stress

DAT: Disposizioni Anticipate di Trattamento

INAIL: Istituto Nazionale Assicurazione Infortuni sul Lavoro

ISS: Istituto Superiore di Sanità

ISTAT: Istituto Nazionale di Statistica

NCCN: National Comprehensive Cancer Network

RSA: Residenza Sanitaria Assistenziale

SARS-CoV-2: Severe Acute Respiratory Syndrome – Coronavirus-2 or Coronavirus Disease 2019

SIPO: Società Italiana di Psico-Oncologia.

Keywords

COVID-19, Health workers, Psychological reactions, Health policy, Oncologists, Cancer, Crisis interventions

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DOI: 10.31038/CST.2020523

Abstract

ACBD3 breast cancer research to date reveals that overexpression at mRNA and protein level is near universal in breast tumour tissue and that high ACBD3 expression is associated with worse patient prognosis. ACBD3 has been shown to have an important role in specifying cell fate and maintaining stem cell pools in neurological development and deletion of ACBD3 in human cell lines prevents cell division. Combined with observations that β-catenin expression and activity is increased when ACBD3 is overexpressed it has been hypothesised that ACBD3 promotes breast cancer by increasing Wnt signalling. This may only be one aspect of ACBD3’s effects as its expression and localisation regulatessteroidogenesis, calcium mediated redox stress and inflammation, glucose import and PI(4)P production which are all intrinsically linked to breast cancer dynamics. Given the wide scope for a role of ACBD3 in breast cancer, we explore its interactors and the implications of preventing these interactions.

Keywords

ACBD3, Breast cancer, Chromosome 1, Golgi, NUMB, PI4Kβ, Phosphatidylinositol, Protein kinase A, Steroidogenesis, Wnt signalling, 1q

Introduction

Although breast cancer incidence has increased in recent years, largely due to improved diagnostic techniques, greater awareness and the introduction of national screening programmes, mortality rates are declining as result of earlier detection and improved treatment regimes. Despite this, treating advanced disease remains difficult and there is a need to identify new therapeutic targets. Proteins encoded by the q-arm of chromosome 1 are of particular interest as regions of 1q are frequently amplified and overexpressed in breast cancer leading to the hypothesis that 1q is important in disease development and progression [1-3]. The frequency at which regions of arm 1q were amplified was investigated and the 1q42.12 locus was found to be amplified in both breast cancer cell lines and primary tumours with a number of genes in or near this region also being over expressed [4]. Of emerging interest is ACBD3, a Golgi protein with multiple functions, only recently linked to breast cancer [5].

ACBD3 was discovered as an interactor of GOLGB1 and named GCP60, and independently discovered as aninteractor of the mitochondrial translocator protein TSPO and protein kinase A and named PAP7 [6,7]. Having found that each of these names were describing one aspect of a diverse adapter protein it was renamed by the HUGO gene nomenclature committee in 2004 as Acetyl CoA Binding Domain containing protein 3, or ACBD3, reflecting its functional groups and protein family rather than any particular role, of which there are many (www.genenames.org). In addition to the acyl CoA binding domain at its N-terminus, ACBD3 contains a Golgi dynamics (GOLD) and a glutamine rich Q domain as well as a proline rich region (Figure 1) [8]. The GOLD domain is found in Golgi and lipid trafficking proteins and makes up the C-terminus of ACBD3 (aa384-526). It is a β-strandrich domain and is responsible for ACBD3 localization to the Golgi via direct interaction with GOLGB1 [6]. ACBD3 is a largely unstructured or loosely structured protein, as many linkers are, and of all the recognisable domains only the GOLD domain structure has been solved by X-ray crystallography with the rest of ACBD3 being modelled by NMR and predictive modelling software (Figure 1). The Q domain is a glutamine rich region (aa241-308) which forms a long loop made of alpha helices [8]. The N-terminal ACB domain binds Acyl CoA and Palmitoyl CoA in other ACBD family proteins but the function of this domain in ACBD3 is unclear. To the N-terminus of the ACB domain is a proline rich region (aa21-60), which is indicative of protein-protein interaction sites and may complement the ACB domain whichis often found paired with protein-protein interaction domains such as the Pleckstrin homology domain (PH) and the Src homology domain in other proteins. ACBD3 makes essential interactions with an unusually high number of protein partners in cellular processes as diverse as Golgi structure, steroid synthesis and glucose import; other functions not reviewed here include iron transport and a causal role in Huntington’s disease progression [7,9-12]. ACBD3 is essential for neural development and human cell lines do not divide when ACBD3 is excised by CRISPR-CAS9 [13].

Figure 1. Predicted 3D structure of human ACBD3.The structure is modelled by Phyre2 software using the primary amino acid sequence which agrees strongly with crystal structures of individual ACBD3 domains and related proteins [86]. From the N-terminus in blue to the C-terminus in red ACBD3 clearly contains 3 domains: the ACBP domain, the Q domain and the Golgi dynamics (GOLD domain) respectively connected by flexible linkers with an N terminal proline rich region. The hydrophilic surface of acbd3 has been superimposed on ACBD3 showing the electrostatic charge of the protein model with red depicting negative charge and blue depicting positive charge.

ACBD3 in Breast Cancer

The q arm of Chromosome 1 contains many genes important in cancer progression or tumour suppression: NRAS, JUN, MYCL, ESRRG, ARF1 and RAB25are amongst the best known. There are however many more 1q genes that are amplified in breast cancer with deletions strikingly rare despite common deletions in the p arm. Some of these genes (PI4Kβ, PIP5K1A and HIST2H2BE) have more recently been recognised as oncogenic with ACBD3 being the latest 1q gene observed to affect breast cancer [14]. ACBD3 mRNA is reported to be upregulated in breast tumour tissue matched against adjacent normal tissue in all subtypes [5]. Protein levels of ACBD3 were upregulated in 8 breast cancer cell lines (MDA-MB453, MDA-MB-415, BT549, MDA-MB-231, ZR-75-30, SKBR3, T47D and MCF7) compared to 2 normal breast epithelial cell samples (NBEC1 and NBEC2). In a cohort of Chinese breast cancer patients ACBD3 protein expression increased as cancer stage became more advanced. Kaplan-Meier survival curves were plotted and it was found that high levels of ACBD3 mRNA in breast tumour tissue predicted lower rates of patient survival and that this made a large difference in stage III and IV cancers with 60% probability of survival at 120 months when ACBD3 expression is low but less than 30% probability of survival when ACBD3 expression is high.

The ACBD3 containing 1q42.12 locus is seen to be amplified in an additional 6 breast cell lines (BRCAMZ01, BT20, HCC2218, MDAMB436, SUM149, ZR751) and 6 out of 25 primary breast tumours in a breast cancer 1q amplification study [4]. Loss of region 1q42.12 was seen in only 1 cell line (UACC812) where the terminal ~38 megabases of arm 1q were deleted and loss of 1q42.12 was not observed in any primary tumour samples. RNA expression levels revealed that 1q42.12 is located in the middle of a region of gain coined G7, the largest region of gain (in bases) on chromosome 1 in breast cancers.Overexpression of ACBD3 in cell cultures caused increased side populations of stemlike cancer cells and inhibition of ACBD3 by siRNAsignificantly reduced these populations [5]. GSEA analysis found that CTNNB1- and TCF4-activated gene signatures both positively correlated with ACBD3 expression [5]. CTNNB1 encodes the β-catenin protein which, in response to Wnt signalling, accumulates in the cytoplasm and then translocates to the nucleus where it propagates the Wnt signal.ACBD3 overexpression led to an increase of β-catenin in the cytoplasm and nucleus compared to when ACBD3 expression was low (65% versus 20% nuclear and cytoplasmic localisation) [5]. TCF4 is a transcription factor for genes that code proteins in the Wnt signalling pathway. When TCF4 was knocked down, the self-renewal ability of ACBD3-expressing cells was abolished suggesting that ACBD3 may promote cancer stem cell propagation via the Wnt/ β-catenin signalling pathway in breast cancer.All of this provides strong evidence that ACBD3 overexpression affects breast cancer but the ACBD3 protein has many binding partners, in disparate cellular pathways and cells appear to have few redundancies for the essential roles of ACBD3.

ACBD3 and Steroidogenesis

Although often considered a resident Golgi protein due its structural role and interactions with other structural components, ACBD3 can also be found at other membranes including the cytosolic cell membrane and at the outer mitochondrial membrane (OMM). ACBD3 interacts withtranslocator protein TSPO (previously the peripheral-type benzodiazepine receptor) on the cytosolic OMM and stimulates cholesterol transport from the OMM to the IMM (inner mitochondrial membrane) (Figure 2) [7,15]. P450scc (CYP11A1) makes direct contact with the IMM and converts cholesterol to pregnenolone, the precursor to mammalian steroids, by side chain cleavage [16,17]. TSPO is anchored to the voltage dependent anion channel VDAC1 and makes up approximately 2% of OMM proteins. TSPO tethers cytosolic ACBD3 at the OMM and ACBD3 subsequently recruits protein kinase A (PKA) via the PKARIα subunit. This brings PKA into proximity with one of its substrates, the steroidogenic acute regulatory (StAR) protein which is phosphorylated on residues S57 and S195 by PKA [18]. StAR then facilitates cholesterol import from the OMM to the IMM, the rate limiting step in steroidogenesis. ACBD3 overexpression increases chorionic gonadotropin-induced steroid production; increased steroid production has obvious implications for cancer progression by enabling self-sufficiency in growth signals, a hallmark of cancer [19,20].

Figure 2. ACBD3 in redox stress and steroidogenesis. ACBD3 is a Golgi resident protein but also has functions elsewhere in the cell. At the Outer Mitochondrial membrane (OMM) ACBD3 is essential for mediating interactions between PKA holoenzyme (via direct tethering with the PKAR1α subunit shown) and two of its substrates: StAR and VDAC1.
a) The phosphorylation state of StAR determines whether cholesterol can cross the IMM and be converted to pregnenolone, the basic building block of all steroid hormones in mammals. Cholesterol import is the rate limiting step in steroidogenesis and ACBD3 is indispensable for this process [18].
b) VDAC1 is a Ca²⁺ ion import channel at the OMM and phosphorylation by PKA closes this channel to prevent calcium import. Mitochondrial import of Ca²⁺ forms part of the calcium homeostasis mechanism in the cell, closing the VDAC1 ion channel causes cytosolic Ca²⁺ concentration to rise in the cell which leads to redox stress and local inflammation. Again, ACBD3 is essential for localising PKA to the mitochondria where it can then phosphorylate the VDAC1 substrate [27].

PKAR1α is a tumour suppressor gene and is important in primary pigmented nodular adrenocortical disease (PPNAD) nodule formation and tumorigenesis in mice and humans. Mutation of PKAR1α leads to hypercortisolism that drives tumorigenesis, and high ACBD3 expression in steroidogenic tissues (of which the adrenal cortex is one) may contribute to the overexpression/over activity of the mutant PKAR1α [21]. PPNADs are characterised by a resistance to apoptosis which in itself contributes to cancer occurrence and is another hallmark of cancer [20]. The first publication to suggest any link between ACBD3 and cancer demonstrated that ACBD3 follows the same expression profile as PKAR1α in PPNAD tissue and speculated that, in tumorigenesis, this could lead to deregulation of steroid synthesis [21]. More recent studies have shown that PKA activation may instead have a suppressive effect on cancer [22,23], whilst others show PKARIα is upregulated in cancer cell lines [24,25].

ACBD3 in Redox Stress

In a separate process, glutamate induces expression of TSPOand increased TSPO recruits ACBD3 and PKA to the mitochondria. Glutamate is a signalling molecule that is known to cause acute neurotoxicity [26-28]. PKA phosphorylates the calcium channel protein VDAC1, preventing Ca²⁺ import into the mitochondria (Figure 2). This causes Ca²⁺ accumulation in the cytosol which signals redox stress via the calcium sensing CamKII and its effector NADPH oxidase (NOX5) leading to inflammation by increased reactive oxygen species (ROS). Glutamate mediated redox stress is particularly important in neuro-inflammationwhere TSPO is not expressed in healthy brain tissue but can accumulate in age related degenerative disease or after traumatic stress, leading to increased ACBD3 mitochondrial recruitment and subsequent VDAC1 phosphorylation by PKA which may contribute to neurodegeneration [29]. VDAC1 is important in Ca²⁺ homeostasis, especially mitochondrial Ca²⁺ homeostasis which controls the metabolism of mitochondria and therefore energy availability in the cell [30]. Dysregulation of cellular energetics is a hallmark of cancer and an inflammatory environment can be tumour promoting when chronic and over time [20].

ACBD3 and Insulin Mediated Glucose Import

GLUT4 (glucose transporter type 4) allows the facilitated diffusion of glucose from the surroundings into cells,andis sequestered into storage vesicles (GSVs) that are tethered to Golgi membranes by TUG (Tether containing UBX domain for GLUT4), Golgin-160 and ACBD3 when insulin is absent (Figure 3) [31]. ACBD3-bound TUG can be acetylated on lysine 549 which has a higher binding affinity with Golgin-160 than with ACBD3 [10]. In response to insulin receptor activation the cytoplasmic effector of insulin PIST (PDZ interacting specifically with TC10) binds Golgin-160 and catalyses the cleavage of acetylated TUG. This releases GSVs allowing them to fuse with the plasma membrane where GLUT4 forms a channel for glucose import [32]. GLUT4 is continuously cycled away from plasma membranes back into GSVs to increase the on/off response of insulin sensitive cells when insulin is not present. GLUT4 exocytosis is regulated by tankyrase 1 as are several other ACBD3 related processes including Golgin45 expression and the promotion of β-catenin transcription in the Wnt signalling pathway [33,34].

Figure 3. The effect of insulin on TUG, the interaction between TUG and ACBD3, and the recycling of GLUT4 storage vesicles to regulate glucose import [10,31].
a) 1. Extracellular insulin binds the transmembrane insulin receptor (IR) causing receptor activation. 2. The active IR tyrosine phosphorylates CBL inside the cell. 3. Phosphorylated CBL recruits the CRK-C3G complex to the membrane lipid raft sub domain facilitating interaction of C3G and TC10α. 4. C3G activates TC10α which subsequently activates its effector: PIST. 5. PIST relocates to the Golgi causing the release of GSVs with embedded GLUT4 transporter which fuse with the cell membrane, 6. allowing glucose to enter the cell. GLUT4 is continuously cycled away from the membrane in GSVs creating a fast on and off switch for insulin dependent glucose import.
b) 1. ACBD3 interacts with TUG and is dependent on the acetylation state of TUG. 2. Acetylation of TUG on lysine 549 causes TUG to preferentially bind Golgin-160 over ACBD3. 3. PIST, activated by the insulin receptor signalling cascade, also binds Golgin-160 and catalyses the cleavage of acetylated TUG causing GSVs to be released into the cytoplasm 4. to fuse with the cell membrane. 5. GLUT4 is continuously cycled away from the cell membrane embedded in GSVs and is sequestered back to the Golgi where they bind TUG.

The hormone 17β-oestradiolhas a central role in breast cancer progression, ithas been found to upregulateGLUT4 expression and translocation to the membranein breast cancer cell lines and was associated with increased glucose uptake [35-37]. GLUT4 is being investigated as a target for breast cancer therapy as part of an informed approach to target the Warburg effect. Downregulation of GLUT4 by siRNAimpairs viability of MDA-MB-231 and MCF7 breast cancer cell lines and increases mitochondrial oxidation of pyruvate [38]. The EGFR/HER2-targeted drug lapatinib has been shown to downregulate GLUT4 in ER-/HER²⁺ HMEC cell lines, and GLUT4 downregulation by siRNA in these cell lines led to the formation of normal acini structures in 3D culture [39].The insulin receptor (IR) is upregulated in breast cancer and is a potential target for breast cancer therapy as it has been demonstrated that knock down of IR by shRNA and inhibition by peptide drugs inhibits breast cancer cell growth [40-43].

ACBD3, PI4Kβand Phosphatidylinositols

Phosphatidylinositol 4 Kinase III beta (PI4Kβ) is a lipid kinase that converts phosphatidylinositol (PI) into phosphatidylinositol 4-phosphate (PI(4)P) [44]. PI4Kβ is localised to the Golgi by extension of an amphipathic helix at the N-terminus of PI4Kβ (aa44-64) through the Q domain alpha helices loop of ACBD3 (aa241-308) [8]. The small GTPase Rab11 binds PI4Kβ to support this interaction whilst ACBD3 also interacts with GOLGB1 on the Golgi surface bringing PI4Kβ in close and constant contact withits PI substrate embedded in the lipid bilayer. ACBD3 does not affect the enzymatic activity of PI4Kβ directly by this interaction but, by tethering it to the Golgi membrane, PI4Kβ is proximal to the PI substrate and does not rely on diffusion through the cytoplasm for the phosphorylation of substrate. PI4Kβ is heavily implicated in breast cancers with 20% of primary tumours showing over expression of PI4Kβ at the protein level [45,46]. PI4Kβ is a chromosome 1q gene (at 1q21.3) and is reported to have increased gene copy number in 62% of 939 patient breast tumour samples [14]. Evidence of PI4Kβ upregulation at the protein level in breast ductal carcinoma samples from the human protein atlas was also found by Waugh. Independent of its lipid kinase function, PI4KIIIβ also mediates indirect phosphorylation and activation of AKT (Protein kinase B), an important kinase in breast cancer signalling [46,47]. AKT dysregulation drives many breast cancers by promoting cell cycle progression and suppressing apoptosis, it is commonly overexpressed or constitutively active [47].

Both PI and PI(4)P are cellular signalling molecules and docking sites on the membrane for other proteins including ARF1 (ADP-ribosylation Factor 1), which is essential for the formation of COPI vesicles and Golgi function including localisation of Golgin-160 to the Golgi; ARF1 is encoded by a gene adjacent to ACBD3 on chromosome 1 (1q42.13) [48-50]. PI4Kβ is also positioned on chromosome 1q, where amplification is common in breast cancers and its substrates localise ARF1 to membranes. ACBD3 is hijacked by some picornavirusviral proteins to form replication organelles and recruits PI4Kβ to these sites to enrich them for PI(4)P [51,52]. This is another example of how the role of ACBD3 is contextual and dependent on its cellular location, cell cycle position and binding partners. PI4Kβ has been found to be a target in malaria and drugs to inhibit PI4Kβ have already been developed [53]. There have so far been no publications on PI4Kβ drug inhibition in cancer.

ACBD3 Cell Signalling in Neurogenesis

Mammalian NUMB, an endocytic adapter protein, is involved in cytosolic signalling and is segregated asymmetrically into one daughter cell during the mitosis of neural progenitor cells and inhibits NOTCH [54]. This asymmetric distribution of NUMB results in 1 identical pluripotent daughter cell (high NUMB protein level) to maintain the population of neuronal precursors and 1 differentiated neuron cell (low NUMB protein level). This balances the need to create neurons through NOTCH signalling and maintain the pool of precursor cells in embryonic neurogenesis by NOTCH inhibition (Figure 4) [55,56]. ACBD3 was identified as a NUMB binding partner after observations that ACBD3 cytosolic release during mitosis was paired with NUMB mediated cell fate [57]. The ACBD3 interacting region on NUMB is essential for NUMB activity and interaction with ACBD3 increases NUMB activity [57]. The C-terminus of ACBD3 binds with the N-terminus of NUMB, and NOTCH also binds the N-terminus of NUMB [58]. Cytosolic ACBD3 expression leads to inhibition of NOTCH, suggesting that NOTCH inhibition by NUMB is conserved from drosophila to mammals indicating that ACBD3 and NUMB are both required to specify cell fate in neural progenitors. ACBD3 is bound to Golgi/mitochondrial membranes through most of the cell cycle and can only bind NUMB during mitosis when the breakdown of the Golgi releases ACBD3 into the cytosol (Figure 4). Constitutively cytosolic mutant ACBD3 inhibits neurogenesis in mouse embryos resulting in fewer neurons. This indicates that permanently cytosolic ACBD3 is preventing differentiation in otherwise neuronal fated cells and it achieves this by binding NUMB outside of mitosis [57].

Figure 4. The differential regulation of NOTCH signalling by ACBD3 and NUMB in neurogenesis. Cytosolic NUMB (yellow shading) acts synergistically with cytosolic ACBD3 (blue shading) to inhibit NOTCH signalling (represented by a green nucleus) and specifies progenitor cell fates during mitosis [57]. In one daughter cell, cytosolic ACBD3 binds to NUMB increasing the ability of NUMB to inhibit NOTCH and causing that daughter cell to remain a progenitor like its parent. The other daughter cell does not contain NUMB and so NOTCH signalling cannot be prevented despite the presence of cytosolic ACBD3 protein and so the daughter cell begins to differentiate into a neuron. Paradoxically NUMB quickly accumulates in the differentiating cell but because the Golgi has reformed at this point, ACBD3 is membrane bound and cannot influence NUMB-NOTCH protein interactions, instead NUMB promotes neuron differentiation and survival through other pathways. This leads to the creation of one daughter neuron and one progenitor cell to balance neurodevelopment and stem cell pools.
a) NUMB accumulates asymmetrically in one half of a progenitor cell before mitosis. ACBD3 is bound to the Golgi apparatus and other organelles (not shown) and does not interact with NUMB.
b) During mitosis, the Golgi apparatus fragments into vesicles and ACBD3 is released into the cytosol where it can interact with asymmetrically distributed NUMB.
c) One of the daughter cells will contain NUMB and cytosolic ACBD3. ACBD3 increases the ability of NUMB to bind and inhibit NOTCH. Without NOTCH signalling the daughter cell remains a progenitor cell to maintain the pool of neuronal precursors.
d) The second daughter cell will contain cytosolic ACBD3 but no NUMB protein meaning that NOTCH is not inhibited and enters the nucleus signalling to the cell to differentiate.
e) NUMB protein is produced in the differentiating cell in G1 but at this time the Golgi has reformed and ACBD3 is no longer free in the cytoplasm and cannot interact with NUMB and so cannot inhibit NOTCH signalling.

Krüppel-like factor 9 (KLF9) is a tumour suppressor and is significantly down regulated in invasive breast cancers, endometrial carcinoma, glioblastoma and colorectal cancer, and its expression can inhibit growth of tumour xenografts from glioblastomaneurospheres [59,60].  ACBD3 and NOTCH1 expression are suppressed by KLF9 in endometrial carcinoma cells and both proteins promote breast cancer progression, specifically in cancer stem cell maintenance [5,61,62]. KLF9 supresses glioblastomaderivedneurosphere formation by 60% in controls but only by 33% when NOTCH1 expression is constitutively active strongly suggesting that KLF9 must suppress other proteins relevant to glioblastoma cancer progression and this could include ACBD3 [59]. NOTCH1, NOTCH3 and JAG1expression isassociated with poor survival in breast cancer patients with high NOTCH1 expression conferring a 66% chance of mortality and 74% chance of relapse at 10 years [63]. NOTCH receptorshave been found to have activating mutations in triple negative breast cancers and result in the upregulation of NOTCH controlled genes [64]. NOTCH overexpression was able to transform the MCF10A breast cell line and reduce its sensitivity to apoptotic drugs such as staurosporine, melphalan, or mitoxantrone, and overexpression of NUMB reverted the transformation [61].High levels of NOTCH in breast tumours are significantly associated with nuclear phospho-Erk 1 and 2 conferring an association between NOTCH and Ras-MAPK expression [65]. Inhibition of NOTCH and NOTCH-related proteinshave therefore become a target for therapy [66]. ACBD3 overexpression prevents NOTCH signalling in neurogenesis but this is reliant on NUMB expression and only during mitosis when the Golgi is fragmented. The NOTCH suppressorsNUMB and its paralogue NUMB-L are predictably down regulated in breast cancers and their overexpression reduces epithelial to mesenchymal transition in triple negative breast cancer cell lines [67-69]. NUMB-deficient breast cancer cells have an increased ability to form cancer stem cell pools and NUMBdownregulation causes inactivation of p53 [70,71].

Discussionand Future Perspectives

Throughout this review an argument is presented that ACBD3 may do more than promote Wnt signalling in the context of breast cancer. Dysregulation of cellular energetics, sustaining proliferative signalling, replicative immortality andtumour-promoting inflammation are all hallmarks of cancer and overexpression of ACBD3 could conceivably support any or all of these [10,18,21,27,57]. Other factors including the position of ACBD3 on chromosome 1 in close proximity to other oncogenes, and the number of its binding partners and pathways already being targeted for cancer therapies leave ACBD3 nothing short of overlooked.ARF1 and RAB4 are located close to ACBD3, both at 1q42.13, withinOrsetti’s(4) region of gain G6 and were both found to be significantly overexpressed at the mRNA level in breast cancer. RAB4 in conjunction with RAB5 promotes and drives metastasis by facilitating the formation of invadosomes containing membrane type 1 matrix metalloprotease (MT1-MMP) and β3 integrin which together degrade the extracellular matrix, a process vital for cancer invasion and metastasis [72]. RAB4 is overexpressed in breast cancers and unsurprisingly associated with increased cell motility, it is one of many RAS related proteins that has clinical significance in cancer [73]. ARF1 is the most amplified gene of the ADP-ribosylation factor family in breast cancers and its amplification is associated with increased gene transcription and worse prognosis for patients [74]. ARF1 inhibition prevents metastasis of tumour xenografts in immuno-deficient mice and is replicable in zebrafish models of breast cancer metastasis. ACBD3 proximity to ARF1, RAB4 and other 1q oncogenes may confer a huge selective advantage to breast cancer cells with amplifications of these lociproviding these cells with both survival and invasive advantages.

Tankyrase 1 regulates GLUT4 exocytosis and β-catenintranscription,and ACBD3 interacts with proteins in both pathways [75-78]. Tankyrase 1 controls the expression of Golgin45 which is a direct binding partner of ACBD3 [79]. Tankyrase also targets Axin for degradation leading to increased Wnt signalling, known to be aberrant in breast cancers andis reported to be effected by ACBD3 [5,76,80]. Tankyrase 1 and 2 are currently being targeted as cancer therapeutics because of their interactions inmany carcinogenic pathways [77,81-83]. PI4Kβ expression in breast cancer correlates with poor patient outcomes and its locus (1q21.3) is a biomarker for breast cancer [46,84]. It is most associatedas an ACBD3 binding partner and the ACBD3 interaction has a solved X-ray crystal structure [8]. PI4Kβ mutants that do not bind ACBD3 have been engineered and drugs that inhibit PI4Kβ are available which aids its study [53,85]. ACBD3deletion is embryonic lethal and may be invaluable for normal cell division [12,57]. As it does not have an enzymatic function an inhibitor for ACBD3 may not be viable and non-targeteddownregulation may not be desirable, instead targeting one or more of its protein-protein interactions or partners may be the route to new treatments in breast cancer.

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