Abstract

   Functional oncogenic links between ErbB2 and ERRα in HER2+ breast
   cancer patients support a therapeutic benefit of co-targeted therapies.
   However, ErbB2 and ERRα also play key roles in heart physiology, and
   this approach could pose a potential liability to cardiovascular
   health. Herein, using integrated phosphoproteomic, transcriptomic and
   metabolic profiling, we uncovered molecular mechanisms associated with
   the adverse remodeling of cardiac functions in mice with combined
   attenuation of ErbB2 and ERRα activity. Genetic disruption of both
   effectors results in profound effects on cardiomyocyte architecture,
   inflammatory response and metabolism, the latter leading to a decrease
   in fatty acyl-carnitine species further increasing the reliance on
   glucose as a metabolic fuel, a hallmark of failing hearts. Furthermore,
   integrated omics signatures of ERRα loss-of-function and doxorubicin
   treatment exhibit common features of chemotherapeutic cardiotoxicity.
   These findings thus reveal potential cardiovascular risks in discrete
   combination therapies in the treatment of breast and other cancers.

   Subject terms: Cardiomyopathies, Biochemical networks
     __________________________________________________________________

   Murine hearts deficient in ErbB2 and/or ERRα are used to profile the
   adverse cardiac remodeling associated with potential targeted breast
   cancer treatments by phosphoproteomic, transcriptomic and metabolomic
   profiling.

Introduction

   The heart relentlessly relies on the coordinated regulation of
   mitochondria, energy-producing pathways, and signaling conduits to fuel
   contraction and sustain blood flow, and these functions are dependent
   on the precise control of metabolite levels, gene expression, and
   enzyme activities. Any derangement can negatively impact cellular
   homeostasis and drive pathological remodeling. Heart failure remains an
   important cause of mortality worldwide posing a tremendous burden on
   the healthcare system^[58]1. Characterization of cardiac metabolic,
   molecular, and structural reprogramming events in response to genetic
   or extrinsic factors is key to understanding the metabolic
   flexibilities of the heart, identify causal determinants and risk
   factors, and ultimately guide drug development and treatment strategies
   for a wide range of diseases.

   The tyrosine kinase receptor ErbB2 (also referred to as HER2) is
   well-known for its oncogenic activity in breast cancer (BCa), but it
   also plays an important role in both cardiac embryonic development and
   in the adult heart^[59]2,[60]3. Genetic or pharmacological inhibition
   of ErbB2 has been shown to cause dilated cardiomyopathy (DCM),
   characterized by chamber dilatation and decreased
   contractility^[61]4–[62]6. Treatment regimens for HER2+ BCa typically
   involve ErbB2-targeted therapies including trastuzumab in combination
   with chemotherapies such as the anthracycline doxorubicin and
   alkylating agent cyclophosphamide to enhance the anti-tumor effects of
   HER2-blockade, albeit increasing the cardiotoxic risk from 3-7% to 27%
   of patients^[63]7–[64]9. Impaired stress responses and cardiomyocyte
   apoptosis consequential to compromised cell survival and repair are
   implicated in trastuzumab-induced cardiac dysfunction^[65]10.
   Doxorubicin-induced adverse cardiac effects are the most severe;
   however, both doxorubicin and cyclophosphamide can cause mitochondrial
   damage and dysfunction, oxidative and nitrative stress, calcium
   deregulation, inflammation, and fibrosis^[66]10,[67]11. The precise
   underlying mechanisms for the observed cardiotoxicities remain
   incompletely understood, and inevitably, treatment cessation due to
   adverse cardiac events is undesirable, warranting further investigation
   into the identification of underlying risk factors and causal
   mechanisms.

   Orphan nuclear receptor oestrogen-related receptor α (ERRα, NR3B1) is a
   key transcriptional regulator of mitochondrial function, redox
   homeostasis and energy metabolism^[68]12–[69]14, thus being an
   attractive therapeutic target for the treatment of metabolic disorders
   and diseases including type 2 diabetes, obesity and
   cancer^[70]15–[71]17. ERRα is also a broad regulator of cardiac
   programs including intracellular fuel sensing, fatty acid β-oxidation
   (FAO), citric acid cycle (CAC), ATP transport, and calcium
   handling^[72]18. ERRα is essential for the bioenergetic and functional
   adaptation to cardiac pressure overload induced by transverse aortic
   constriction via its direct transcriptional regulation of ATP
   generating programs^[73]19.

   In malignant BCa, ERRα, and ErbB2 are functionally linked and their
   expression levels correlate positively^[74]20–[75]22. Notably,
   attenuation of ERBB2 signaling disrupts ERRα activity^[76]22, and
   reciprocally, ERRα ablation reduces ERBB2 amplicon gene transcription
   and impedes ErbB2-induced murine BCa development^[77]20. Thus,
   targeting ERRα in combination with ErbB2 may offer a therapeutic
   benefit in HER2+ patients^[78]23. However, as both factors play
   cardioprotective roles, we investigated the consequence of their
   combined loss of function on the heart. Herein, we report that in-depth
   cross-analyses of cardiac phosphoproteomic, transcriptomic and
   metabolic profiles reveal that while ErbB2 and ERRα play distinct and
   complementary roles in maintaining myocardial homeostasis and function,
   combined genetic attenuation of ErbB2 and ERRα severely amplifies
   adverse cardiac remodeling and metabolic inflexibility observed in mice
   deficient in a single factor. Furthermore, an integrated omics
   signature driven by ERRα loss was predictive of doxorubicin response
   and reciprocally, an assembled cardiac multi-omics doxorubicin
   signature was found to be characteristic of decreased ERRα activity,
   identifying a hitherto unsuspected functional link between ERRα and
   doxorubicin action in the heart.

Results

Loss of ErbB2 and ERRα signaling independently contribute to myocardial
dysfunction

   To investigate the functional consequence and possible molecular and
   genetic crosstalk between ErbB2 and ERRα in the adult heart, we crossed
   ERRα knock-out (KO) mice^[79]24 with the ErbB2 hypomorphic mouse
   model^[80]25 (ErbB2 KI) giving rise to ErbB2 KI/ERRα KO mice (herein
   referred to as KI:KO) in a FVB background. KI:KO mice are viable,
   fertile and do not display any gross anatomical abnormalities. The
   concomitant loss of both ErbB2 and ERRα in KI:KO mice was confirmed by
   RT-qPCR (Supplementary Fig. [81]1a). Given that ErbB2 KI mice develop
   age-dependent DCM with earliest signs of pathophysiology at 4-months of
   age^[82]6, cardiac function was evaluated on 15-week-old ErbB2 KI, ERRα
   KO, and KI:KO mice in comparison to WT controls.

   Loss of ErbB2 and ERRα had opposite effects on heart size with KI:KO
   reflecting the average outcome (Fig. [83]1a–c). Masson’s trichrome
   staining revealed increased fibrosis in ERRα KO hearts and to a greater
   extent in KI:KO mice (Fig. [84]1a, d). Ultrasound echocardiography
   confirmed the development of DCM in the ErbB2 KI model, marked by a
   reduction in left ventricular (LV) contractile function as demonstrated
   by lower LV ejection fraction (LVEF) and LV fractional shortening
   (LVFS) parameters as well as increased LV dilatation with augmented
   end-systolic (LVIDs) and end-diastolic dimensions (LVIDd) relative to
   WT (Fig. [85]1e–i). ERRα KO mice also presented with DCM similarly to
   ErbB2 KI mice (Fig. [86]1e–i), with cardiac fibrogenesis likely playing
   a crucial role in the pathogenesis. KI:KO displayed a synergistic
   effect of impaired ErbB2 and ERRα signaling on DCM development
   (Fig. [87]1e–i), not associated with greater vascular defects,
   cardiomyocyte apoptosis or hypertrophy (Supplementary Fig. [88]1b–e).
   Consistent with their increased disease severity, KI:KO hearts
   expressed higher transcript levels of two biomarkers of hemodynamic
   stress and heart failure, atrial natriuretic peptide (ANP) and brain
   natriuretic peptide (BNP)^[89]26 (Fig. [90]1j), thus supporting both
   ERRα- and ErbB2-dependent contributions to the observed DCM.

Fig. 1. Cardiac phenotype of mice with impaired ErbB2 and/or ERRα activity.

   [91]Fig. 1
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   a Representative Hematoxylin and Eosin (H&E), wheat germ agglutinin
   (WGA), and Masson’s trichrome staining of heart sections from 15-week
   old mice with ErbB2 and/or ERRα loss-of-function. Scale bars, 500 μm
   (H&E) and 50 μm (WGA, Trichrome). b, c Mean heart weight (HW) (b) and
   normalized HW to body weight (BW) ratios (c) of mice (n = 30). d
   Quantification of interstitial fibrosis (Trichrome, n = 6). e
   Representative M-mode echocardiographic images for each mouse genotype.
   f–i Percent of left ventricular ejection fraction (LVEF) (f) and LV
   fractional shortening (LVFS) (g) as well as echo-derived LV internal
   diameter end systole (LVIDs) (h) and end diastole (LVIDd) (i) in mice
   (WT and ErbB2 KI, n = 7; ERRα KO and KI:KO, n = 5). Cardiac RT-qPCR
   analysis of the genes encoding atrial natriuretic peptide (ANP) and
   brain natriuretic peptide (BNP), both markers of cardiac dysfunction
   (j) (n = 6). Data are normalized to Rplp0 levels. Data in (b–d) and
   (f–j) represent means ± SEM, *p < 0.05 by ANOVA relative to WT
   controls, unless otherwise indicated. See also Supplementary
   Fig. [93]1.

Cardiac phosphoproteomics reveals ErbB2 and ERRα dependencies for proper
structural and functional integrity

   Changes in protein phosphorylation and associated signaling mechanisms
   have been linked with cardiac dysfunction. Unbiased label-free
   LC-MS/MS-based phosphoproteomics profiling identified 2066
   phosphorylated peptides mapped to 602 proteins using a high confidence
   site localization probability of the modified residues (≥0.7). Relative
   to WT, KI:KO hearts displayed 48 to 63% more significant phosphopeptide
   changes (limma, p < 0.05, |FC | ≥ 1.5) than ErbB2 KI and ERRα KO
   models, respectively, mostly serine modifications, and clustered more
   closely with ErbB2 KI samples (Fig. [94]2a, Supplementary
   Fig. [95]2a–c, and Supplementary Data [96]1). Consolidated
   phosphoserine and phosphothreonine motifs of differentially expressed
   phosphopeptides were marked by a strong preference for proline at
   position +1 and arginine at position −3 in all groups compared to WT
   (Fig. [97]2b and Supplementary Fig. [98]2d). The MoMo tool^[99]27,
   which expands on the robust algorithm Motif-x^[100]28, identified the
   pSP and RXXpS motifs in all 3 models vs WT, the former being the most
   frequently observed motif (Fig. [101]2c and Supplementary
   Fig. [102]2e). Among the other enriched motifs identified, we found the
   pTP motif in both ERRα KO and KI:KO as well as pSXXE and RXXpT motifs
   in the KI:KO model. PhosphoMotif Finder^[103]29 predicted GSK3 and ERK
   family kinases to be responsible for a significant number of altered
   phosphopeptides along with CaMKII, PKA, and PKC (Fig. [104]2c).

Fig. 2. Phosphoproteomics identification of ErbB2 and ERRα post-translational
control of cardiomyocyte structure and metabolism.

   [105]Fig. 2
   [106]Open in a new tab

   a Volcano plots illustrating the significantly up-regulated (red) and
   down-regulated (blue) phosphopeptides from cardiac phosphoproteomics
   profiling of mouse models relative to WT (limma, p < 0.05, |FC | ≥ 1.5,
   n = 5). b Consolidated phosphomotifs generated by PHOSIDA^[107]79 of
   pSer-modified phosphopeptides found differentially expressed in the
   mouse models compared to WT showing site-specific amino acid
   preferences adjacent to the central serine phosphorylated residue. c