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Frontiers in Endocrinology logoLink to Frontiers in Endocrinology
. 2022 Oct 18;13:1010092. doi: 10.3389/fendo.2022.1010092

PKC-mediated phosphorylation and activation of the MEK/ERK pathway as a mechanism of acquired trastuzumab resistance in HER2-positive breast cancer

Jeanesse Scerri 1, Christian Scerri 1, Felix Schäfer-Ruoff 2, Simon Fink 2, Markus Templin 2, Godfrey Grech 3,*
PMCID: PMC9623415  PMID: 36329884

Abstract

Protein expression, activation and stability are regulated through inter-connected signal transduction pathways resulting in specific cellular states. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins (phospho-protein profile) in key signal transduction pathways, in breast cancer cellular models of trastuzumab resistance. To this effect, we utilized a multiplex, bead-based protein assay, DigiWest®, to measure around 100 proteins and protein modifications using specific antibodies. The main advantage of this methodology is the quantification of multiple analytes in one sample, utilising input volumes of a normal western blot. The intrinsically trastuzumab-resistant cell line JIMT-1 showed the largest number of concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β catenin stabilization by inhibitory phosphorylation of GSK3β, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance cellular models. The overexpression of upstream RAS/RAF, however, was confined to JIMT 1; meanwhile, in a cellular model of acquired trastuzumab resistance generated in this study (T15), entry into the ERK pathway seemed to be mostly mediated by PKCα activation. This is a novel observation and merits further investigation that can lead to new therapeutic combinations in HER2-positive breast cancer with acquired therapeutic resistance.

Keywords: acquired resistance, breast cancer, phospho-profile, PKC/MEK/ERK, signalosome, HER2 positive, patient stratification

Introduction

HER2 and trastuzumab

The human epidermal growth factor receptor 2 (HER2) protein is overexpressed in approximately 15% of breast cancers (1). Having no known ligands, it forms heterodimers with other members of the HER family of receptor tyrosine kinases (HER1/EGFR, HER3, HER4 (2). HER2 activation results in the phosphorylation and activation of multiple downstream signaling proteins, including phospholipase C γ1 (PLCγ1), phosphatidylinositol 3-kinase (PI3K) regulatory and catalytic subunits, RasGAP, and heat shock protein 90 (3). The ensuing signaling cascade, mostly represented by the PI3K/AKT and RAS/RAF/ERK pathways, leads to uncontrolled cellular proliferation and invasion. Protein phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase, is also a central regulatory component of PI3K/Akt pathway; its inactivation through phosphorylation at its tyrosine residue p.tyr307 has been found to be increased in HER2-positive tumor samples and correlated to tumor progression (4). Of interest, HER2 signaling increases c-myc phosphorylation at Ser62 and is maintained through attenuation of the phosphatase, PP2A (5). In fact, PP2A activators promote c-myc protein degradation (6). Clinically, high nuclear myc staining is positively associated with lymph-node positive disease in HER2 amplified breast cancer tumors (7). Hence, the HER2-MYC-PP2A axis is of clinical relevance and provides potential therapeutic targeting of breast cancers with co-amplification of HER2 and MYC. In a murine model of HER2 knock-in mammary tumors, overexpression of HER2 significantly upregulated β-catenin and its transcriptional targets Cyclin D1, SOX9 and c-Myc. High cytoplasmic β-catenin, expression of basal markers and loss of membranous E-cadherin are associated with poor prognosis in human HER2+ invasive ductal carcinomas (8).

Trastuzumab (Herceptin®), an immunoglobulin G1 (IgG1) antibody consisting of two mouse-derived antigen binding sites specific to the HER2 receptor extracellular domain (ED) and a humanized Fc portion (9), has been hailed as one of the successes of personalized medicine for the treatment of HER2-positive breast cancer. Its mode of action, though not yet fully understood, involves both direct and indirect pathways of inhibition. The former is brought about by the binding of the antibody to the ED of Her2, inhibiting its cleavage (10), and resulting in downstream signaling inhibition (mainly the PI3K/Akt pathway (11), through internalization and degradation of the HER2 receptor (12). The inhibition of heterodimer formation with other HER family members leads to reduced VEGF-mediated angiogenesis (13). The most important indirect pathway of inhibition is the activation of antibody-dependent cellular toxicity by the recruitment of Fc-competent immune effector cells (14). Trastuzumab is always administered adjuvantly to chemotherapeutic agents, where it also inhibits the repair of chemotherapy-induced DNA damage (15).

Trastuzumab resistance mechanisms

Nonetheless, intrinsic resistance to the drug in some cases, and tumour recurrence due to acquired resistance in others, are important caveats of the targeted therapy (16). Mechanisms of trastuzamab-HER2 binding inhibition are associated with intrinsic resistance. Steric hindrance by cell surface proteins such as mucin-4 (MUC4) inhibits this binding (17); sensitivity to trastuzumab was enhanced upon knockdown of MUC4 expression in a JIMT-1 cell model (18), suggesting that MUC4 occupies the trastuzamab-binding sites of HER2. Overexpression of stem cell marker CD44 and its ligand, hyaluronan, also mask the trastuzumab binding domain on the HER2 ED and provide an independent prognostic factor for poor disease-free survival in HER2 positive patients treated with adjuvant trastuzumab (19). Proteolytic cleavage of the HER2 receptor generates a constitutively activated, truncated HER2 receptor lacking the ED, p95-HER2, which is associated with lymph node involvement (20) and trastuzumab resistance (21), attributed to the absence of the trastuzumab-binding domain.

Deregulation of signalling pathways downstream to HER2, and the activation of alternative cellular proliferation pathways, are alternative trastuzumab resistance mechanisms. Suppressed PTEN phosphatase activity prevents trastuzumab-induced growth arrest through sustained PI3K/AKT phosphorylation and signal transduction (22). A combination of low PTEN expression and PIK3CA oncogenic mutations predict trastuzumab response in HER2-positive breast cancer patients (23). In addition, trastuzumab-induced growth arrest of HER2-positive tumour cells is counteracted by an increase in insulin-like growth factor-1 receptor (IGF-IR) signalling (24). IGF-IR mediated trastuzumab-resistance is attributed to enhanced degradation of p27 and hence release from cell cycle arrest induced by trastuzumab treatment (25). Resistance to trastuzumab was also associated with increased expression of c-Met (26), and CAV-1 involved in caveolae-mediated endocytosis (27).

Immune escape is another mechanism of trastuzumab resistance. Genomic polymorphisms in FcγRIIIa that significantly suppress the affinity of IgG1 antibodies to the immune cell Fcγ receptor will impair ADCC activation (28). Furthermore, exosomes may transfer transforming growth factor beta 1 (TGFβ1), an immunosuppressive cytokine, and programmed death-ligand-1 (PD-L1), a lymphocyte activation inhibitor, to tumour cells. The presence of these exosomes was correlated with resistance to ADCC, suggesting a role of exosomes in suppressed immune-mediated response to trastuzumab (29). Exosomes generated by SKBR3 cell lines are also positive for the receptor, and may act as decoy by binding to trastuzumab, reducing its availability to target tumour cells (30).

High-throughput biomarker detection

In addition to diagnostic biomarkers, the discovery of predictive markers of treatment resistance is a key aspect of personalized medicine. In the era of network medicine and high-throughput “omics”, it is important to study the interplay of the different complex mechanisms leading to drug resistance. The classification of breast cancer into molecular subtypes with prognostic and predictive implications, based on high-throughput gene expression data, has led to the development of gene panels such as the Oncotype DX (31) or the MammaPrint™ (32) assays. For Her2-positive breast cancer, however, there is no FDA-approved gene panel to date for the clinical prediction of response to trastuzumab-containing treatment regimes. The use of bead-based, multiplex RNA (33) and protein (34) assays has shown effectiveness in medium- to high-throughput cancer biomarker discovery and detection.

This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins in key signal transduction pathways, in cellular models of resistance. We utilized JIMT-1 as a cellular model of intrinsic resistance, and generated an acquired trastuzumab resistance model (T15) to study differential signaling signatures.

Materials and methods

Generation of trastuzumab-resistant cell line

SKBR3 cells with acquired trastuzumab resistance were obtained by conditioning with the drug as described by Zazo et al. (35). Briefly, the cell line (ATCC® HTB-30™), grown in Dulbecco’s Modified Eagle Medium (DMEMM, Sigma-Aldrich, St. Louis, MO) supplemented with 10% foetal bovine serum (FBS) and 1% GlutaMAX™ (Thermo Fisher Scientific, Waltham, MA), was acclimatised for 30 days in 10µg/mL trastuzumab followed by long-term culturing in medium containing 15µg/mL of the drug. Resistance to trastuzumab was confirmed by cell viability assay (MTT), which showed a maintenance of ≥80% viability after 72 hours incubation with 25-100µg/mL trastuzumab concentration (compared to the parent cells which showed reduced viability at these drug concentrations). The resulting cell line will be henceforth referred to as T15. The JIMT-1 cell line (DSMZ ACC-589), kindly donated by M. Barok at the University of Helsinki, Finland, was cultured in DMEM supplemented with 10% heat-inactivated FBS.

Bead-based, multiplex phosphoprotein profiling

High-throughput multiplex phosphoprotein profiling was subsequently carried out by the DigiWest® technique, as described by Treindl et al. (36), on the parental and conditioned cell lines. Briefly, cell pellets containing 5x105 cells or more were lysed, and gel electrophoresis and blotting onto PVDF membranes was performed using the NuPAGE system as recommended by the manufacturer (Life Technologies, Carlsbad, CA, USA). The membranes were washed in PBST, then incubated in NHS-PEG12-Biotin (50μM) in PBST for 1 hour to biotinylate the blotted proteins, followed by another wash in PBST and drying. Individual sample lanes were cut into 96 molecular weight fractions (0.5mm each), with the separated proteins in each fraction eluted in 96-well plates using 10μL elution buffer (8M urea, 1% Triton-X100 in 100mM Tris-HCl pH 9.5) per well. The eluted proteins from each molecular weight fraction were then coupled with neutravidin-coated Luminex beads (MagPlex, Luminex, Austin, TX, USA) of a specific bead identity (red-infrared spectral wavelength), yielding 96 size-specific bead identities per sample. 384 Luminex bead sets were employed and the protein-loaded beads from 4 different sample lanes were pooled into a bead-mix having a concentration of 40 beads/µL in carboxy block storage buffer (CBS), which was sufficient for over 100 antibody incubations. Antibodies specific proteins and phosphoproteins with roles in HER2 downstream signaling pathways and other aforementioned mechanisms of interest were utilized ( Table 1 ).

Table 1.

Selected antibodies, fluorescence intensities and Log2 FC in protein & phosphoprotein quantities in T15 and JIMT-1 relative to SKBR3.

Fluorescence Intensity LOG2 FC rel. to SKBR3
Pathway Analyte Supplier Cat. No. Species + Clonality JIMT1 SKBR3 T15 JIMT1 T15
PI3K/mTOR 4E-BP1 Epitomics 1557-1 Rb mAb 2490 240 283 3.37 0.24
PI3K/mTOR 4E-BP1 - phosphoThr70 Cell Signaling 9455 Rb pAb 754 119 164 2.66 0.46
PI3K Akt Cell Signaling 4685 Rb mAb 3567 2125 2901 0.75 0.45
PI3K Akt1 Cell Signaling 2938 Rb mAb 1105 1443 2341 -0.39 0.70
PI3K Akt1 - phosphoSer129 Cell Signaling 13461 Rb mAb 1 1 328 0.00 8.36
mTOR AMPK alpha Cell Signaling 2532 Rb pAb 657 418 544 0.65 0.38
mTOR AMPK alpha - phosphoThr172 Cell Signaling 2535 Rb mAb 1217 135 300 3.17 1.15
MEK/ERK A-Raf Cell Signaling 4432 Rb pAb 2952 1177 1051 1.33 -0.16
MEK/ERK A-Raf - phosphoTyr301/Tyr302_58kDa Biorbyt orb5910 Rb pAb 28345 31881 29893 -0.17 -0.09
MEK/ERK A-Raf - phosphoTyr301/Tyr302_68kDa Biorbyt orb5910 Rb pAb 21540 22850 19923 -0.09 -0.20
MEK/ERK A-Raf - phosphoTyr301/Tyr302_Total Biorbyt orb5910 Rb pAb 49885 54729 49815 -0.13 -0.14
PI3K/WNT beta-Catenin Cell Signaling 8480 Rb mAb 24084 188 382 7.00 1.02
PI3K/WNT beta-Catenin - phosphoSer552 Cell Signaling 9566 Rb pAb 449 1 1 8.81 0.00
PI3K/WNT beta-Catenin (non-pospho Ser33/37/Thr41; active) Cell Signaling 8814 Rb mAb 3541 1 1 11.79 0.00
MEK/ERK b-Raf - phosphoSer445 Cell Signaling 2696 Rb pAb 208 143 160 0.54 0.16
Cell cycle CDK4 Cell Signaling 12790 Rb mAb 32451 3328 2931 3.29 -0.18
PI3K c-myc_57kDa Cell Signaling 9402 Rb pAb 207 205 241 0.01 0.23
PI3K c-myc_70kDa Cell Signaling 9402 Rb pAb 549 520 337 0.08 -0.63
PI3K c-myc_Total Cell Signaling 9402 Rb pAb 756 724 577 0.06 -0.33
MEK/ERK c-Raf Cell Signaling 9422 Rb pAb 632 161 135 1.97 -0.25
MEK/ERK c-Raf - phosphoSer259 Cell Signaling 9421 Rb pAb 2423 873 874 1.47 0.00
MEK/ERK c-Raf - phosphoSer289/296/301 Cell Signaling 9431 Rb pAb 374 186 168 1.01 -0.15
PI3K CREB - phosphoSer133 Cell Signaling 9198 Rb mAb 177 1 56 7.47 5.80
PI3K eIF4E Cell Signaling 2067 Rb mAb 13776 16186 17424 -0.23 0.11
PI3K eIF4E - phosphoSer209 Cell Signaling 9741 Rb pAb 348 927 1193 -1.41 0.36
MEK/ERK Elk-1 Cell Signaling 9182 Rb pAb 653 656 813 -0.01 0.31
MEK/ERK Elk-1 - phosphoSer383 Cell Signaling 9186 ms mab 644 1580 1651 -1.29 0.06
MEK/ERK Erk1/2 (MAPK p44/42)_p42 Cell Signaling 4695 Rb mAb 17917 30972 41263 -0.79 0.41
MEK/ERK Erk1/2 (MAPK p44/42)_p44 Cell Signaling 4695 Rb mAb 3100 1702 1774 0.87 0.06
MEK/ERK Erk1/2 (MAPK p44/42)_Total Cell Signaling 4695 Rb mAb 21016 32673 43036 -0.64 0.40
MEK/ERK Erk1/2 (MAPK p44/42) - phosphoThr202/Tyr204_p42 Cell Signaling 4370 Rb mAb 4211 788 1190 2.42 0.60
MEK/ERK Erk1/2 (MAPK p44/42) - phosphoThr202/Tyr204_p44 Cell Signaling 4370 Rb mAb 1656 93 294 4.16 1.67
MEK/ERK Erk1/2 (MAPK p44/42) - phosphoThr202/Tyr204_Total Cell Signaling 4370 Rb mAb 5866 880 1482 2.74 0.75
MEK/ERK ERK1/2 (MAPK) - phosphoThr202/Tyr204_p42 Cell Signaling 9101 Rb pAb 4653 257 480 4.18 0.90
MEK/ERK ERK1/2 (MAPK) - phosphoThr202/Tyr204_p44 Cell Signaling 9102 Rb pAb 1340 113 143 3.57 0.34
MEK/ERK ERK1/2 (MAPK) - phosphoThr202/Tyr204_Total Cell Signaling 9103 Rb pAb 5993 368 621 4.02 0.75
MEK/ERK Erk2 (MAPK p42) Cell Signaling 9108 Rb pAb 2649 6720 10792 -1.34 0.68
WNT GSK-3 alpha Cell Signaling 4337 Rb mAb 4395 4403 4928 0.00 0.16
WNT GSK3 alpha - phosphoSer21_51kDa Cell Signaling 9331 Rb pAb 229 443 409 -0.95 -0.11
PI3K/WNT GSK3 alpha/beta - phosphoSer21/Ser9_Total Cell Signaling 9331 Rb pAb 548 443 409 0.31 -0.11
PI3K GSK3 beta - phosphoTyr216_47kDa Abcam ab68476 Rb mAb 129 232 281 -0.85 0.28
PI3K GSK3 alpha - phosphoTyr279_51kDa Abcam ab68476 Rb mAb 706 125 148 2.50 0.25
PI3K GSK3 alpha/beta - phosphoTyr279/Tyr216_Total Abcam ab68476 Rb mAb 834 355 429 1.23 0.27
PI3K GSK3 beta - phosphoSer9 Cell Signaling 9336 Rb pAb 511 1 1 9.00 0.00
PI3K GSK3 beta Cell Signaling 9315 Rb mAb 11035 3642 2050 1.60 -0.83
HER2 Her2 DAKO A0485 Rb pAb 3595 5008 8862 -0.48 0.82
Multiple HSP 90 Abcam ab59459 Ms mAb 150805 456009 861935 -1.60 0.92
IGF1 IGF1 receptor beta (Insulin receptor beta, CD221) Cell Signaling 3018 Rb mAb 308 153 166 1.02 0.12
MEK/ERK MAPKAPK-2 Cell Signaling 12155 Rb mAb 392 556 453 -0.50 -0.29
MEK/ERK MEK 1 Cell Signaling 9124 Rb pAb 1128 610 644 0.89 0.08
MEK/ERK MEK1 - phosphoSer298 Cell Signaling 98195 Rb mAb 896 1 1 9.81 0.00
MEK/ERK MEK1 - phosphoThr292 Cell Signaling 26975 Rb mAb 1036 1 1 10.02 0.00
MEK/ERK MEK1/2 - phosphoSer217/Ser221 Cell Signaling 9154 Rb mAb 3200 135 416 4.56 1.62
MEK/ERK MEK2 Cell Signaling 9125 Rb pAb 953 171 131 2.48 -0.38
MEK/ERK Mnk1 Cell Signaling 2195 Rb mAb 239 161 164 0.57 0.03
MEK/ERK MSK1 - phosphoSer376 Millipore 04-384 Rb mAb 2302 2436 9259 -0.08 1.93
PI3K/mTOR mTOR (FRAP) Cell Signaling 2983 Rb mAb 3077 1394 2232 1.14 0.68
PI3K/mTOR mTor - phosphoSer2448 Cell Signaling 5536 Rb mAb 1211 521 997 1.22 0.94
MEK/ERK p38 MAPK Cell Signaling 9212 Rb pAb 572 258 273 1.15 0.08
Cell cycle p53 R&D af1355 Gt pAb 9935 1601 2117 2.63 0.40
PI3K/mTOR p70 S6 kinase Cell Signaling 2708 Rb mAb 5905 2365 3004 1.32 0.34
PI3K/mTOR p70 S6 kinase - phosphoThr421/Ser424 Cell Signaling 9204 Rb pAb 632 93 177 2.76 0.92
PI3K PDK1 Cell Signaling 3062 Rb pAb 1398 808 1294 0.79 0.68
PI3K PDK1 - phosphoSer241 Cell Signaling 3061 Rb pAb 142 73 193 0.96 1.39
PI3K PI3-kinase p110 delta_110kDa Santa cruz sc-7176 Rb pAb 734 220 238 1.74 0.11
PI3K PI3-kinase delta_60kDa Santa cruz sc-7176 Rb pAb 11463 12042 11384 -0.07 -0.08
PI3K PI3-kinase delta_Total Santa cruz sc-7176 Rb pAb 12196 12262 11620 -0.01 -0.08
PI3K PI3-kinase p110 alpha Cell Signaling 4255 Rb pAb 31 253 266 -3.05 0.07
PI3K PI3-kinase p110 beta_110kDa Millipore 04-400 Rb mAb 2897 916 995 1.66 0.12
PI3K PI3-kinase p110 beta_60kDa Millipore 04-400 Rb mAb 1514 1835 1665 -0.28 -0.14
PI3K PI3-kinase p110 beta_Total Millipore 04-400 Rb mAb 4409 2750 2659 0.68 -0.05
PI3K PI3-kinase p85 alpha Epitomics 1675-1 Rb mAb 363 87 118 2.06 0.44
PI3K PI3-kinase p85 Cell Signaling 4292 Rb pAb 437 129 157 1.76 0.28
PI3K PI3-kinase p85/p55 - phosphoTyr458/Tyr199_55kDa only Cell Signaling 4228 Rb pAb 336 3158 3154 -3.23 0.00
PI3K PKC (pan) - phosphoSer660 Cell Signaling 9371 Rb pAb 1073 1180 4669 -0.14 1.98
PI3K PKC (pan) gamma - phosphoThr514_80kDa Cell Signaling 38938 Rb mAb 1102 1141 2533 -0.05 1.15
PI3K PKC (pan) gamma - phosphoThr514_85kDa Cell Signaling 38938 Rb mAb 2613 3150 5891 -0.27 0.90
PI3K PKC (pan) gamma - phosphoThr514_Total Cell Signaling 38938 Rb mAb 3715 4290 8423 -0.21 0.97
PI3K PKC alpha - phosphoSer657 Abcam AB180848 Rb mAb 1769 1122 4227 0.66 1.91
PI3K PKC alpha - phosphoThr497 Abcam AB76016 Rb mAb 1526 1948 2841 -0.35 0.54
PI3K PKC alpha BD Biosciences 610107 Ms mAb 1 77 80 -6.27 0.05
PI3K PKC alpha/beta II - phosphoThr638/Thr641 Cell Signaling 9375 Rb pAb 980 1570 1473 -0.68 -0.09
PI3K PP2A C Cell Signaling 2259 Rb mAb 5653 3171 2571 0.83 -0.30
PI3K PP2A C - phosphoTyr307 R&D AF3989 Rb pAb 4986 20990 10084 -2.07 -1.06
PI3K PTEN Cell Signaling 9552 Rb pAb 228 203 304 0.16 0.58
MEK/ERK Ras Cell Signaling 8955 Rb mAb 2280 742 1103 1.62 0.57
Cell cycle Rb Cell Signaling 9309 Ms mAb 405 131 119 1.62 -0.15
Cell cycle Rb - phosphoSer795 Cell Signaling 9301 Rb pAb 149 53 1 1.49 -5.73
Cell cycle Rb - phosphoSer807/Ser811 Epitomics 2004-1 Rb mAb 1618 342 347 2.24 0.02
Multiple RSK 1 (p90RSK) Cell Signaling 9344 Rb pAb 1038 306 334 1.76 0.13
Multiple RSK 1 (p90RSK) - phosphoSer380 Cell Signaling 9341 Rb pAb 317 228 853 0.48 1.90
Multiple RSK 1 (p90RSK) - phosphoThr573 Abcam ab62324 Rb mAb 556 115 303 2.27 1.40
Multiple RSK 1/2/3 Cell Signaling 9347 Rb pAb 1059 438 453 1.27 0.05
Multiple RSK 3 Epitomics 2012-1 Rb mAb 1005 409 648 1.30 0.67
Multiple RSK 3 - phosphoThr356/Ser360 Cell Signaling 9348 Rb pAb 87 1 104 6.45 6.70
PI3K/mTOR S6 ribosomal protein Cell Signaling 2317 Ms mAb 6810 14092 11270 -1.05 -0.32
PI3K/mTOR S6 ribosomal protein - phosphoSer235/Ser236 Cell Signaling 2211 Rb pAb 11823 31835 20663 -1.43 -0.62
PI3K/mTOR S6 ribosomal protein - phosphoSer240/Ser244 Cell Signaling 2215 Rb pAb 9573 38584 21969 -2.01 -0.81
PI3K/mTOR TSC2 (Tuberin) Cell Signaling 4308 Rb mAb 2138 489 1073 2.13 1.13
PI3K/mTOR Tuberin/TSC2 - phosphoSer1387 Cell Signaling 23402 Rb mAb 1369 233 564 2.55 1.27

Antibodies were organized into the main canonical pathways of signal transduction and cellular proliferation. Antibody species: Rb: rabbit, Ms: mouse, Gt: goat; antibody clonality: mAb: monoclonal, pAb: polyclonal. Fluorescence intensity values less than 100 are deemed inaccurate and should be interpreted with caution. Fold changes ≥1 are denoted in light orange and fold changes ≤ -1 are denoted in light green.

For each target protein or phosphoprotein to be quantified, an aliquot of the DigiWest bead-mixes was added to a well of a 96-well plate containing 50μL assay buffer (Blocking Reagent for ELISA supplemented with 0.2% milk powder, 0.05% Tween-20, and 0.02% sodium azide, Roche). Following a brief incubation in assay buffer, the buffer was discarded by keeping the 96-well plate on a magnet. The beads were then incubated with 30µL of a specific primary antibody diluted in assay buffer per well. After overnight incubation at 15°C on a shaker, the bead-mixes were washed twice with PBST and PE-labelled (Phycoerythrin) secondary antibodies (Dianova) specific to the primary antibody species were added and incubated for 1 hour at 23°C. Beads were washed twice and resuspended in PBST prior to the readout on a Luminex® FlexMAP 3D®.

For the quantification of the antibody specific signals, the DigiWest® analysis tool (version 3.8.6.1, Excel-based) was employed. This tool uses the 96 values for each initial lane obtained from the Luminex® measurements on the 96 molecular weight fractions, identifies the peaks at the appropriate molecular weight, calculates a baseline using the local background, and integrates the peaks. The obtained values are based on relative fluorescence (AFI, accumulated fluorescence intensity). For analysis, the data was normalized to the total protein amount corresponding to the sample, and the relative quantification of each protein and phosphoprotein was expressed as log2 fold-change (FC) in T15 and JIMT-1 as compared to SKBR3. Differentially expressed targets were organized into established signal transduction pathways and phosphosite log2 FC were used to predict whether each protein was under- or over-activated.

Results and discussion

MEK/ERK pathway is a central mechanism of acquired trastuzumab resistance

Phosphoinositide-dependent kinase-1 (PDK1) activity was significantly increased (log2 FC(PDK1) = +0.7; log2 FC(pPDK1ser241 = +1.4) in T15. A lack of significant change in RAF expression was expected to be consistent with a lack of alteration in downstream MEK1/2 signaling; however, the MEK/ERK pathway was still found to be overall activated. The expression of total MEK1 was equivalent, while that of MEK2 was slightly downregulated (log2 FC = -0.36) in T15 when compared to SKBR3. Meanwhile, activated pMEK1ser217/221/pMEK2ser222/226 (antibody does not distinguish between the two isoforms) was significantly upregulated in T15 (log2 FC = +1.6). ERK (MAPK) activity reflected the changes observed in its upstream activator, MEK: despite minimal changes in total protein expression (log2 FC(ERK1) = +0.06, log2 FC(ERK2) = +0.41), phosphorylated (active) forms of ERK1 and ERK2 were over-represented, thus resulting in a higher ratio of phosphorylated to total ERK1/2 (log2 FC(pERK1thr202/tyr204 = +1.7; log2 FC(pERK2thr185/tyr187 = +0.6). The results were confirmed with two different antibody clones (Cell Signaling product ID 4370 and 9101; log2 fold changes reported here obtained with the former), both of which bind to ERK1 and ERK2 and give two specific peaks of 44 and 42 kDa, respectively ( Figure 1 ; Table 1 ). T15 also showed hyper-activation of the ribosomal protein S6 kinase α-5 protein, MSK1 (log2 FC(pMSK1ser376) = +1.9; Figure 1 ). MSK1 is directly phosphorylated by MAPKs at serine 360, threonine 581, and threonine 700, and subsequently autophosphorylates at serine 376 for protein activation (37). Seemingly conflicting roles for MSK1 in breast cancer have been described: it shows tumor suppressor functions by acting as a transcriptional coactivator of P53 and mediating phosphorylation of histone H3 in the transcriptional activation of p21 (37), but has also been associated with epithelial-mesenchymal transition (EMT) and subsequent skeletal metastasis by histone H3 acetylation and phosphorylation of Snail, which downregulates E-cadherin to promote cellular migration and invasion (38).

Figure 1.

Figure 1

Pathways involved in the induced trastuzumab resistance of T15 (log2 FC normalized to SKBR3 signals). Similarly expressed protein: light grey; antibody not available/poor performance: no fill; overexpressed total protein ≥ 1.5-fold (log2 FC ≥ 0.58): light orange; ≥ 2-fold: *; over-represented phosphosite ≥ 1.5-fold: orange; ≥ 2-fold: *; predicted active protein: dark red outline.

Activation of the MEK/ERK pathway is through PKCα activation in acquired resistance

In the absence of RAF overexpression, entry into the MEK/ERK pathway can be mediated by the protein kinase C (PKC) family, via PDK1. PKCα and PKCγ are both members of the diacylglycerol (DAG) sensitive, Ca2+ responsive conventional PKC (cPKC) isoform subgroup. Activation downstream to receptor tyrosine kinases, such as ErbB receptors, involves the Ca2+ sensitive recruitment of phosphatidylinositol (4, 5-bisphosphate [PtdIns (4, 5)P2]-specific phospholipases Cγ1/2 (PtdIns-PLCγ1/2) through their SH2 domains; PDK1-dependent activation loop phosphorylation, together with C-terminal phosphorylations events, catalyze PKC activity by maintaining the active conformation of the kinase domains (39). While PKCγ is more specific to the brain, PKCα is detected in all normal and most tumor tissue types (40). The presence of activated pan-PKC and specifically PKCα was determined by the over-representation of phospho-proteins in T15 (log2 FC(PKCA) = +0.05; log2 FC(pPKCAthr497) = +0.54; log2 FC(pPKCAser657) = +1.91), as well as the overexpression of PDK-1 p-ser241, an autophosphorylation site essential for PDK1 activity ( Figure 1 ). Increased levels of this phosphoprotein are a frequent event in breast cancer metastasis, and have been proposed as a candidate for chemosensitisation in innate and acquired resistance (41).

PKC-α, like other protein kinases, plays a role in the regulation of various cellular functions, ranging from cell proliferation and differentiation to control of apoptosis. Requiring HSP90 (log2 FC in T15 = 0.92) and mTORC2 complex to prime phosphorylation, it is sequentially phosphorylated at Thr497 in the kinase domain by PDK1 and at Thr638 and Ser657 autophosphorylation sites. While in the cytoplasm, the phosphorylated PKC-α is still inactive, until it is recruited to the plasma membrane, where it exerts its functions (42). Its importance in cellular proliferation renders its abnormal expression a transformative event: initial recognition of the role of PKC-α in tumorigenesis was reported by Ways and colleagues (43), where ectopic expression of the isoform in MCF7 cells led to a more aggressive phenotype characterized by increased cell proliferation, anchorage-independent growth, loss of epithelial morphology, and enhanced tumorigenicity in nude mice. Using the same cell line, Gupta et al. (44) attributed the increase in cellular proliferation to ERK activation by PKC-α.

PKC family members were also identified as kinases involved in HER2 endocytosis by Bailey and colleagues (45), by using tanespimycin to inactivate HSP90 (and thus promote receptor internalization for degradation), followed by a kinase inhibitor screen to identify kinases whose inhibition correlated with reduced cell surface clearance of HER2. The activation of PKC by phorbol myristate acetate (PMA), and the specific ectopic expression of constitutively active PKC-α, promoted its co-localization with HER2 into a juxtanuclear compartment without subsequent degradation. Conversely, knockdown of PKC-α by siRNA impaired HER2 trafficking to the ERC. In a previous study, PKC-α was implicated in the positive regulation of cell surface HER2 receptor levels, as assessed by flow cytometry, in breast cancer cell lines classified as HER2 2+ on immunohistochemistry without gene amplification as determined by fluorescence in situ hybridization (FISH) (46).

Mulitple PKC-independent pathways are activated in intrinsic resistance model, JIMT-1

Upon phosphoprotein profiling of JIMT-1 as a HER2-positive breast cancer cell line with intrinsic trastuzumab resistance, it was immediately evident that multiple cell survival and proliferation pathways were simultaneously upregulated in comparison with SKBR3, but these did not involve PKC proteins ( Figure 2 ). Specifically, the RAS/RAF/MEK/ERK pathway was highly activated, together with the overexpression of the highly important kinases, PI3K class Ia (p110β isoform; log2 FC = +1.7) and PDK1 (log2 FC = +0.8). Upregulated cell cycle progression was indicated by the highly over-expressed CDK4 (log2 FC: +3.3) and the overall downregulation of the retinoblastoma-associated protein (Rb) tumor suppressor (log2 FC(Rb) = +1.6; log2 FC(pRbser807/811 = +2.2; normalized AFI(pRbser795) = 189 (not detected in SKBR3)). GSK3β activity was suppressed (log2 FC(GSK3β) = +1.6; AFI(pGSK3βser9) = 511 (not detected in SKBR3)), leading to increased expression (log2 FC: +7.0) and activity (non-phospho-ser33/37/thr41: AFI = 3541; not detected in SKBR3) of β-catenin, which is associated with an increase in transcriptional activation. Enhanced cell survival was indicated by the overall activation of the cAMP-response-element-binding protein (CREB); despite total protein expression being below the cutoff in all cell lines, the active phosphosite at ser133 was not expressed in SKBR3 but expressed (normalized AFI = 177) in JIMT-1 ( Figure 2 ; Table 1 ).

Figure 2.

Figure 2

Pathways involved in JIMT-1 trastuzumab resistance (log2 FC normalized to SKBR3 signals). AFISimilarly expressed protein: light grey; antibody not available/poor performance: no fill; overexpressed total protein ≥ 1.5-fold (log2 FC ≥ 0.58): light orange; ≥ 2-fold: *; under-expressed total protein ≤0.67 (log2 FC ≤ -0.58): light green; ≤ 0.5-fold: *; overexpressed phosphosite ≥ 1.5-fold: orange; ≥ 2-fold: *; underexpressed phosphosite ≤ 0.67: green; ≤ 0.5-fold: *; predicted active protein: dark red outline; predicted inactive protein: dark blue outline.

Control of these complex signal transduction cascades by feedback loop mechanisms makes the interpretation of some phospho-proteomic results more challenging. Specifically, both activators of the S6 ribosomal protein (RPS6), the p70S6 kinase (p70S6K/S6K1) and the ribosomal S6 kinase (p90RSK/RSK1), were activated in both models of resistance (i.e. T15 and JIMT-1), while RPS6 itself was downregulated in both cell lines. Activation of p70S6K was confirmed by the over-representation of its phosphorylation target on mTOR at serine 2448 (47), while activation of RSK1 was confirmed by the over-representation of different activating phosphosites, in both models ( Figures 1 , 2 : Table 1 ). Also of interest, deregulation of PP2A and the HER2-MYC-PP2A axis were not apparently involved in the intrinsic resistance of JIMT-1 to trastuzumab or the resistance acquired by T15. The PP2A C regulatory subunit was overexpressed at a log2 FC of 0.83 in JIMT-1 and was not significantly differentially expressed in T15, while its inactivating phosphosite p.tyr307 was significantly underexpressed in both cell lines. Meanwhile, no change in expression of c-myc was observed in the two cell lines in relation to SKBR3 ( Table 1 ).

Clinical perspectives

In this study, we focused on the differential protein expression and phosphorylation events in a cellular model of intrinsic resistance (JIMT-1) and one with generated trastuzumab-induced acquired resistance (T15). PKC-mediated MEK/ERK pathway activation was observed in the acquired model (T15) only. Apart from its above-mentioned functions, PKC-α expression maintains the invasiveness of triple-negative breast cancer (TNBC) and endocrine resistant cell lines through upregulation of FOXC2, a transcriptional repressor of p120-catenin (CTNND1); a high FOXC2:CTNND1ratio was also associated with shorter disease free survival in TNBC patients in The Cancer Genome Atlas (TCGA) dataset (48). FOXC2 is an epithelial-mesenchymal transition (EMT) marker, a process known to be significantly associated with HER2-positive, metastatic breast cancer in the clinical setting (49). Cells undergoing EMT commonly show upregulation of metalloproteinases (50, 51), which promote HER2 cleavage/shedding and thus a high ratio of p95:p185 HER2, associated with trastuzumab resistance and poor disease-free survival in HER2+ breast cancer (52). Assessment of the p95:p185 HER2 ratio in plasma exosomes derived from HER2-positive breast cancer patients (30) is a potential tool for the detection of early metastatic disease and monitoring of response to trastuzumab therapy.

Using the DigiWest® methodology, we interrogated major signal transduction pathways to understand the complex interplay of these pathways and changes following resistance to therapy. Bead-based, multiplex (phospho)protein assays are a very efficient means of studying these pathways, whereby the supporting data from many members of the same pathway, rather than a few candidates (as is permitted by traditional Western blotting techniques) lends robustness to the overall observations. The use of this methodology to characterise exosomes for HER2 receptor ratios, FOXC2 and other EMT markers, metalloproteases, TGFβ; and PD-L1, and other markers of therapeutic resistance can accompany the other developments in liquid biopsy, such as circulating tumour cells (CTCs) (53) and patterns of cell-free nucleic acids in plasma (54), as well as protein biomarkers in other biofluids such as tears (55), to predict disease development and progression. The potential use of DigiWest® to quantitate proteins from various sources provides a multiplex method that can be translated to the clinic, since ultra-high throughput proteomics by mass spectroscopy remain challenging to use in the clinical setting. Understanding treatment resistance mechanisms and incorporating multiplex assays in personalised medicine allows the prediction of early therapeutic resistance and prevents the use of non-beneficial therapies.

Conclusion

MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance cellular models. PKC-mediated MEK/ERK pathway activation in the cellular model of acquired trastuzumab resistance generated in this study (T15) was not observed in the intrinsic model, JIMT-1, which is in turn characterized by the PKC-independent activation of various pathways, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β catenin stabilization by inhibitory phosphorylation of GSK3β, cell cycle progression by Rb suppression, and CREB-mediated cell survival. This is a novel observation which merits further investigation that can lead to new therapeutic combinations in HER2-poitive breast cancer with acquired therapeutic resistance to trastuzumab.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Author contributions

JS carried out the experiments and data analysis and contributed to the draft of the manuscript. FS-R and SF supervised JS during DigiWest analysis that was performed at the NMI institute under the approval of MT. The data analysis was performed using tools provided by MT. CS and GG conceived the study, designed and coordinated the project. GG contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This study was funded by the ALIVE Charity Foundation through the Research Innovation and Development Trust (RIDT), providing a scholarship to JS and bench fees. This work received financial support from the State Ministry of Baden-Wuerttemberg for Economic Affairs, Labour and Tourism.

Acknowledgments

We would like to acknowledge the Institute of Molecular Medicine & Biobanking and the Faculty of Medicine& Surgery for the support in the use of the facilities at the University of Malta.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.


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