The antibody–drug conjugate trastuzumab-emtansine (T-DM1) offers an additional treatment option for patients with HER2-amplified tumors. However, primary and acquired resistance is a limiting factor in a significant subset of patients. Hypoxia, a hallmark of cancer, regulates the trafficking of several receptor proteins with potential implications for tumor targeting. Here, we have investigated how hypoxic conditions may regulate T-DM1 treatment efficacy in breast cancer. The therapeutic effect of T-DM1 and its metabolites was evaluated in conjunction with biochemical, flow cytometry, and high-resolution imaging studies to elucidate the functional and mechanistic aspects of hypoxic regulation. HER2 and caveolin-1 expression was investigated in a well-annotated breast cancer cohort. We find that hypoxia fosters relative resistance to T-DM1 in HER2+ cells (SKBR3 and BT474). This effect was not a result of deregulated HER2 expression or resistance to emtansine and its metabolites. Instead, we show that hypoxia-induced translocation of caveolin-1 from cytoplasmic vesicles to the plasma membrane contributes to deficient trastuzumab internalization and T-DM1 chemosensitivity. Caveolin-1 depletion mimicked the hypoxic situation, indicating that vesicular caveolin-1 is indispensable for trastuzumab uptake and T-DM1 cytotoxicity. In vitro studies suggested that HER2 and caveolin-1 are not coregulated, which was supported by IHC analysis in patient tumors. We find that phosphorylation-deficient caveolin-1 inhibits trastuzumab internalization and T-DM1 cytotoxicity, suggesting a specific role for caveolin-1 phosphorylation in HER2 trafficking.

Implications:

Together, our data for the first time identify hypoxic regulation of caveolin-1 as a resistance mechanism to T-DM1 with potential implications for individualized treatment of breast cancer.

This article is featured in Highlights of This Issue, p. 515

The HER2 gene is amplified in approximately 15% of all patients with breast cancer and is characterized by a relatively aggressive phenotype (1–4). However, a growing arsenal of HER2-targeting drugs has significantly improved the outcome of this patient subgroup (5). Trastuzumab-emtansine (T-DM1) is a HER2-targeted antibody–drug conjugate (ADC) where the cytotoxic microtubule-inhibitory agent DM1 is conjugated to trastuzumab antibody via a stable thioether linker (6). T-DM1 was approved as a single agent for the treatment of patients with locally advanced or metastatic HER2+ breast cancer by the FDA in 2013 (7), and recently showed improved survival as an adjuvant in patients with residual invasive disease after neoadjuvant therapy (8). Moreover, T-DM1 has recently shown promising results in HER2+ lung cancer (9). However, despite its significant clinical efficacy, intrinsic and acquired resistance is a major limiting factor for the therapeutic efficacy of T-DM1 (10–16).

Malignant tumors display regions of severe hypoxia that is associated with resistance to conventional oncologic treatments (chemo- and radiotherapy; refs. 17–19). A far less-explored area is how hypoxia may contribute to tumor cell escape from targeted therapies, such as T-DM1. HER2 is known to heterodimerize with EGFR, and hypoxia was shown to induce constitutive EGFR receptor signaling by delayed sorting to and deactivation in the endolysosomal compartment (20). Moreover, hypoxia may augment ligand-independent EGFR signaling by increased dimerization and prolonged activation in caveolin-1 (CAV1)–associated membrane domains, further leading to enhanced tumor cell proliferation and invasiveness (21). Hypoxia was shown to downregulate membrane protein internalization through a mechanism that involved CAV1 (22), and other studies suggest that HER2 homodimers codistribute with cholesterol-rich membrane raft domains and an involvement of caveolae-dependent mechanisms in the regulation of HER2 trafficking (23–28). Importantly, the possible link between hypoxia, HER2 internalization, and T-DM1 resistance remains unexplored.

Here, we were interested in investigating how adaptive responses to tumor hypoxia conditions may regulate T-DM1 treatment efficacy with potential implications in the management of HER2+ breast cancer.

Cell lines

HER2+ breast cancer cell lines (SKBR3 and BT474) were newly purchased from the ATCC and routinely cultured in HyClone McCoy 5a and DMEM (GE Healthcare Life Sciences), respectively, supplemented with 10% FBS, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich; growth medium). HeLa cells were purchased from the ATCC and routinely cultured in DMEM supplemented with 10% FBS, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (PEST). For cell authentication, the ATCC uses morphology, karyotyping, and PCR-based approaches to confirm the identity of cell lines and to rule out both intra- and interspecies contamination [cytochrome C oxidase I (COI) analysis and short tandem repeat profiling, respectively]. All cells were grown in a humidified 5% CO2 incubator at 37°C and regularly (at least once per month) tested for mycoplasma by DAPI staining and confocal fluorescence microscopy. For all hypoxia-related experiments, cells were grown in hypoxia preconditioned media in a humidified Sci-tive NN Hypoxia workstation (Ruskinn Technology Ltd.) set at 5% CO2, 1% O2, and 37°C.

Breast cancer patient cohort

The patients are participants of The Breast Cancer and Blood Study (BC-Blood Study), which is an ongoing population-based study at the Skåne University Hospital in Lund, Sweden, since 2002 (29). The present tumor microarray (TMA) cohort is based on consecutive patients included in the study between November 2005 and June 2012. Among 789 included patients, 731 were diagnosed with invasive breast cancer. Patients with preoperative treatment (n = 20), distant metastasis within 0.3 years from inclusion (n = 6), and patients with carcinoma in situ (n = 32) were excluded from the study. In total, tumors from 635 patients were evaluated for CAV1 expression by IHC (see below). The study was approved by the Lund University Ethics Committee (Dnr LU75-02, LU37-08, LU658-09, LU58-12, LU379-12, LU227-13, LU277-15, and LU458-15) and performed according to the ethical permit guidelines. All patients signed written informed consent.

Cell transfections and FACS sorting

HeLa CAV1 knockdown (KD) and scrambled control cells, as previously described (22), were transfected with HER2 as per the manufacturer's instructions. Briefly, cells were transfected with 10 μg of HER2 WT plasmid DNA (#16257, Addgene) using lipofectamine 2000 (Thermo Fisher Scientific) transfection cocktail. To obtain HeLa CAV1 KD and scrambled control cells expressing equal levels of HER2, cells were sorted for HER2 expression by FACS. Following incubation with trastuzumab (50 μg/mL; Roche) at 4°C for 30 minutes, cells were washed and incubated with AlexaFluor488-conjugated anti-human antibody (25 μg/mL; #A11013, Thermo Fisher Scientific) in serum-free DMEM at 4°C for another 45 minutes. Cells were washed and resuspended in PBS containing 1% BSA at a cell density of 1 × 106 cells/mL and sorted by FACSAria IIu (BD Biosciences). Sorted cells were collected in selection media (3.5 μg/mL puromycin, 2 mg/mL G418, 1% PEST, and 1% l-glutamine) initially supplemented with 50% FBS for cell recovery, and then with 10% FBS for further use.

For transient, siRNA-mediated CAV1 KD, SKBR3 cells were transfected with 25 nmol/L CAV1 siRNA ON-TARGET plus SMARTpool (L-003467-00-0005/L-003467-00-0010/L-003467-00-0020/L-003467-00-0050, Dharmacon) using DharmaFECT transfection solution. Cells were cultured for 48 hours without media change before further use. For generation of stable pCAV1 mutants, SKBR3 cells were transfected with 2 μg of wild-type CAV1-RFP (WT-CAV1-RFP, control), or a CAV1 tyrosine phosphorylation mimic (Y14D-CAV1-RFP), or phosphorylation-deficient CAV1 (Y14F-CAV1-RFP) plasmids, as previously described (30), using lipofectamine 2000 (Thermo Fisher Scientific) transfection cocktail. Cells were cultured for 24 hours without media change before selection in growth medium supplemented with 50 μg/mL kanamycin.

Cell viability and apoptosis assays

Normoxic or hypoxic SKBR3, BT474, MDAMB468, and HeLa cells, or the various SKBR3 and HeLa cell transfectants, were treated with T-DM1 (Roche AB), trastuzumab, DM1, MCC-DM1, or LMCC-DM1 with a range of concentrations, as indicated in the respective figure legend. Alternatively, SKBR3 cells were treated with mouse anti-HER2 antibody (1:100; clone CB11, Thermo Fisher Scientific) complexed with anti-mouse IgG Fc-monomethyl auristatin F (αMFc-CL-MMAF) secondary ADC (#AM-102-AF, Moradec LLC) or with paclitaxel-albumin nanoparticles (Abraxane), at the indicated concentrations. Following an incubation for 48 to 72 hours at normoxia or hypoxia, MTS reagent (CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit, #G3580, Promega) was added for 3 hours, and the absorbance was measured at 490 nm using VERSAmax tunable microplate reader with SoftMax Pro Software. The cell viability of the compounds was expressed as relative IC50 after normalizing to appropriate controls (only trastuzumab, PBS, or DMSO). Control absorbance values subtracted from the respective baseline absorbance values (medium + PBS or DMSO) were defined as 100% cell viability (100% value), and cells treated with the highest concentration of test compound were defined as 0% cell viability (0% value) control. Relative IC50 values were calculated from Log10 versus normalized response curves (variable slope) equation, generated using GraphPad Prism software v6 (GraphPad Software Inc.).

The Caspase Glo 3/7 cell viability assay was performed as per the manufacturer's instructions. Briefly, normoxic and hypoxic SKBR3 cells were treated with trastuzumab or T-DM1, as indicated, for 72 hours, and then transferred to room temperature (RT) before addition of 100 μL of Caspase-GloR 3/7 reagent (#G8090, Promega) for 2 hours before measuring luminescence using a FLUOstar OPTIMA (BMG LABTECH).

Cell lysate, coimmunoprecipitation, and immunoblotting

Normoxic or hypoxic cells were lysed with nondenaturing lysis buffer [20 mmol/L Tris-HCl, pH 7.4, 137 mmol/L NaCl, 2 mmol/L EDTA, 10% Glycerol, 1% Triton X-100, protease inhibitors (add fresh)] for 20 minutes at 4°C. Cell lysate was clarified by centrifugation at 18,000 × g for 10 minutes at 4°C, and the soluble supernatant was used for further analysis. For immunoprecipitation, cell lysates were swirled with anti-HER2 (#134182, Abcam) or anti-CAV1 antibody (#2941, Abcam) at 4°C overnight. The antibody–antigen solution was mixed with Protein G–conjugated Dynabeads (#10007D, Thermo Fisher Scientific) for 3 hours at 4°C followed by extensive washing using a DynaMag-2 magnetic separator (Thermo Fisher Scientific). Protein G–conjugated Dynabeads and an isotype-matched IgG antibody were used as negative control. Bound proteins were eluted according to the manufacturer's recommendation, mixed with NuPAGE LDS Sample Buffer 4 × (Thermo Fisher Scientific), and heated at 80°C for 10 minutes. Equal amount of proteins was loaded and separated in a NuPage 4–12% Bis-Tris gel (Thermo Fisher Scientific) at reducing conditions, and then transferred onto a polyvinylidene fluoride membrane (Immobilon-FL, Merck KGaA), followed by blocking in TBS 0.05% Tween 20 (TBST) containing 3% skim milk at RT for 1 hour. To probe for CAV1 and HER2, the membrane was incubated with the following antibodies in TBST containing 3% BSA overnight at 4°C: Rabbit anti-CAV1 (1:4,000; #ab2910, Abcam), mouse anti-HER2 (1:1,000; #OP39 Calbiochem, Sigma-Aldrich), and anti–β-actin (1:5,000; #ab8227, Abcam). After washing, the membrane was incubated with horseradish peroxidase–conjugated anti-rabbit (1:10,000; #7074, Cell Signaling Technology) or anti-mouse IgG (1:10,000; #A9044, Sigma-Aldrich) antibodies. Protein bands were visualized by Pierce Enhanced Chemiluminescence Western Blotting Substrate (Thermo Fisher Scientific).

Immunofluorescence and immunohistochemistry

Normoxic or hypoxic SKBR3 and BT474 cells were incubated with trastuzumab (40 μg/mL) for 30 minutes at 4°C (surface) or for the indicated time periods at 37°C (internalization). To visualize only internalized trastuzumab, cell surface–bound trastuzumab was removed with detachment solution (2 mol/L Urea/50 mmol/L Glycine/150 mmol/L NaCl, pH 2.4, 3 × 5 minutes). Subsequently, cells were fixed with 4% PFA for 10 minutes and permeabilized with 0.5% saponin for 15 minutes at RT, and then incubated with AlexaFluor488-conjugated goat anti-human secondary antibody (25 μg/mL) for 1 hour. Nuclei were counterstained with Hoechst 33342 (1:20,000; #1399, Thermo Fisher Scientific) for 10 minutes at RT, and cells were analyzed using Zeiss LSM 710 confocal scanning equipment with Plan-Apochromat 20x/0.8 or a C-Apochromat 63x/1.2 W Korr objective and Zen software (Carl Zeiss). Super resolution imaging details were acquired using the Airyscan detector system (Carl Zeiss).

TMAs were constructed as previously described (29), with duplicate cores of 1.0 mm from each primary tumor using a semiautomated tissue array instrument (Beecher Instruments). For IHC, 4 μm TMA sections were incubated at 60°C for 2 hours, deparaffinized, and then hydrated and pretreated with automatic PT-link system [DAKO, EnVision FLEX + Mouse (LINKER) # K8021] to unmask the epitopes. Sections were then stained for CAV1 with a primary rabbit polyclonal anti-CAV1 antibody (1:1,000; ab2910, Abcam) in Flex TRS low bluffer (pH 6.1) at 96°C for 20 minutes on Indalo P-block and labeled with Polymer EnVision FLEX/HRP to visualize CAV1, and then counterstained with hematoxylin (EnVision FLEX Hematoxylin). CAV1 was scored according to the intensity of cytoplasmic staining of invasive breast cancer cells across the two tumor cores for each patient. If at least 20% of the invasive cells were stained, the intensity was assigned 1 (weak staining), 2 (moderate), or 3 (strong). If less than 20% of the tumor cells were stained, the tumor was assessed as 0 (no staining). Scoring was performed by two independent readers (M. Barbachowska and V. Indira Chandran), and in case of disagreement, a senior evaluator was consulted (B. Nodin), and consensus was reached. All evaluators were blinded to data pertaining to the tumor samples. Nine patients had bilateral invasive breast cancer. For 5 of these patients, CAV1 expression was available for both tumors, and in only one case, CAV1 intensity differed and the value from the side with the highest intensity was used. The tumor characteristics from the evaluated tumor were used in all analyses where tumor characteristics were included.

FACS quantification

Normoxic or hypoxic SKBR3 cells were incubated with trastuzumab (40 μg/mL) for 30 minutes on ice (surface) or 2 hours at 37°C (internalized). For quantifying internalized trastuzumab, cell surface–bound trastuzumab was removed with detachment solution (2 mol/L Urea/50 mmol/L Glycine/150 mmol/L NaCl, pH 2.4, 3 × 5 minutes), followed by blocking of residual surface trastuzumab with unlabeled goat anti-human antibody (50 μg/mL) for 30 minutes. Subsequently, cells were detached, fixed with 4% PFA for 10 minutes, and permeabilized with 0.5% Saponin for 15 minutes at RT. Cells were then incubated with AlexaFluor488-conjugated secondary antibody (25 μg/mL) for 1 hour at RT before FACS analysis using Accuri C6 (BD Biosciences).

Sucrose gradient subcellular fractionation

Subcellular membrane fractionation was performed using a modification of a detergent-free method (31). Normoxic or hypoxic SKBR3 cells were scraped into lysis buffer (150 mmol/L Na2CO3, pH 11, containing 1 mmol/L EDTA, protease inhibitor mixture) and sonicated with 3 cycles of 20-second bursts (QSonica Q125 sonicator). For discontinuous sucrose gradients, equal volume of membrane homogenate was mixed with 80% sucrose in 25 mmol/L MES (2-(N-Morpholino)ethanesulfonic acid sodium salt) and 150 mmol/L NaCl (MBS, pH 6.5) to form 40% sucrose bottom layer, with the above layer of 6 mL of 35% sucrose in MBS, followed by 4 mL of 5% sucrose. The gradient mixture was centrifuged at 175,000 × g (33,000 rpm) for 3 hours at 4°C using a SW41Ti rotor (Beckman Instruments). Samples were removed in 1 mL fractions from the bottom of the tube using a fraction collector. Total protein from 1 mL of each fraction was precipitated with methanol/chloroform, and the final pellet was air-dried and resuspended in RIPA buffer for immunoblotting.

Statistical analyses

Statistical analyses of quantitative experimental methodologies were performed using unpaired Student t test with the GraphPad prism suite. Data are presented as mean ± SD. As for clinical samples, CAV1 expression was analyzed for associations with clinicopathologic characteristics by the χ2 test and linear-by-linear with one degree of freedom, using SPSS Statistics version 22 (IBM Corp., 2013). All P values were two-tailed, and P < 0.05 were considered to be statistically significant.

Hypoxia confers resistance to T-DM1 in HER2+ breast cancer cells

We set out to determine the cytotoxic potency of T-DM1 in breast cancer cells grown in normoxia or hypoxia (1% O2). Normoxic HER2+ cells, as expected, were highly sensitive to T-DM1 at low nanomolar concentrations (IC50, 2.9 and 6.8 nmol/L in SKBR3 and BT474 cells, respectively; Fig. 1A and B), whereas HER2 cells (MDAMB468) were inherently resistant even at the highest concentration tested (10 nmol/L; Fig. 1C). Interestingly, HER2+ cells seemed to acquire relative resistance to T-DM1 under hypoxic conditions (IC50 not reached, NR; Fig. 1A and B), as further supported by decreased caspase 3/7 induction by T-DM1 in hypoxic compared with normoxic cells (Supplementary Fig. S1). To understand if the observed resistance under hypoxia is restricted to T-DM1, we next tested a different anti-HER2 antibody precomplexed with a secondary antibody–monomethyl auristatin F toxin conjugate. Remarkably, hypoxic SKBR3 cells were relatively resistant also to this ADC (IC50, NR), whereas normoxic cells were sensitive at low nanomolar concentration (IC50, 4.7 nmol/L; Fig. 1D). Consistent with previous studies (6), trastuzumab antibody alone caused limited cell death and did not reach IC50 even at the highest concentration of 50 nmol/L, independently of the oxygenation status (Fig. 1E).

Figure 1.

Hypoxia confers resistance to T-DM1 in HER2+ breast cancer cells. Dose–response curves showing T-DM1 sensitivity of SKBR3 (A), BT474 (B), and MDAMB468 (C) cells preincubated in normoxia or hypoxia for 20 hours and then treated with T-DM1 for a further 72 hours in normoxia or hypoxia. Data were normalized for cells treated with trastuzumab (Control, 100%). D, Sensitivity of normoxic or hypoxic SKBR3 cells treated with anti-HER2 precomplexed with toxin-conjugated IgG (αMFc-CL-MMAF) for 72 hours. Data were normalized for αMFc-CL-MMAF alone (Control, 100%). E,In vitro sensitivity of normoxic or hypoxic SKBR3 cells treated with trastuzumab for 72 hours. A–E, Normoxia (black curves) and hypoxia (red curves). Cell viability was measured by MTS assay and is expressed as percentage of control. Data shown are representative from at least three independent experiments, each performed in triplicates. NR, not reached. F, Normoxic or hypoxic SKBR3 cells treated with or without trastuzumab for 2 hours were analyzed by immunoblotting for total p-Akt and p-ERK1/2, and β-actin as loading control. Shown is a representative blot from two independent experiments.

Figure 1.

Hypoxia confers resistance to T-DM1 in HER2+ breast cancer cells. Dose–response curves showing T-DM1 sensitivity of SKBR3 (A), BT474 (B), and MDAMB468 (C) cells preincubated in normoxia or hypoxia for 20 hours and then treated with T-DM1 for a further 72 hours in normoxia or hypoxia. Data were normalized for cells treated with trastuzumab (Control, 100%). D, Sensitivity of normoxic or hypoxic SKBR3 cells treated with anti-HER2 precomplexed with toxin-conjugated IgG (αMFc-CL-MMAF) for 72 hours. Data were normalized for αMFc-CL-MMAF alone (Control, 100%). E,In vitro sensitivity of normoxic or hypoxic SKBR3 cells treated with trastuzumab for 72 hours. A–E, Normoxia (black curves) and hypoxia (red curves). Cell viability was measured by MTS assay and is expressed as percentage of control. Data shown are representative from at least three independent experiments, each performed in triplicates. NR, not reached. F, Normoxic or hypoxic SKBR3 cells treated with or without trastuzumab for 2 hours were analyzed by immunoblotting for total p-Akt and p-ERK1/2, and β-actin as loading control. Shown is a representative blot from two independent experiments.

Close modal

The tumor-inhibiting activity of trastuzumab has been attributed to attenuation of protumorigenic cell signaling downstream of HER2. To further explore if hypoxia-induced resistance to T-DM1 involved hypoxic regulation of HER2-dependent cell signaling activation, the phosphorylation status of multiple kinases was visualized by antibody array, revealing no major differences between normoxic and hypoxic cells under these conditions (Supplementary Fig. S2). Moreover, AKT and ERK phosphorylation inhibition by trastuzumab treatment was similar in normoxic and hypoxic cells (Fig. 1F). We conclude that hypoxia induces resistance specifically to T-DM1, whereas the effect of trastuzumab alone appears insensitive to hypoxic conditions.

Cytotoxicity of T-DM1 catabolites is not altered by hypoxia

We next hypothesized that the relative resistance to T-DM1 under hypoxia was due to reduced efficacy of the DM1 cytotoxin moiety. To this end, we tested active free DM1, linker attached DM1 (MCC-DM1), and lysine-linker attached DM1 (LMCC-DM1) for cytotoxic effects in normoxia and hypoxia. DM1 was toxic both in normoxia and hypoxia in SKBR3 (IC50 9.1 and 7.0 nmol/L, respectively) and BT474 cells (IC50 0.43 and 0.27 nmol/L, respectively; Supplementary Fig. S3A and S3D). MCC-DM1 and LMCC-DM1 carry a net positive charge, which compromises their ability to readily cross the plasma membrane (32, 33). Accordingly, MCC-DM1 and LMCC-DM1 were relatively nontoxic to SKBR3 and BT474 cells (i.e., IC50 NR) both at hypoxic and normoxic conditions (Supplementary Fig. S3B–S3C and S3E–S3F). These findings suggest that hypoxia induces resistance to the intact T-DM1 conjugate and not against its cytotoxin catabolites.

Attenuated trastuzumab internalization at tumor hypoxia conditions

We next investigated the possibility that hypoxic conditions inhibit T-DM1 cytotoxicity through regulation of HER2 expression. Total HER2 as well as constitutive cell surface HER2 expression was unaltered by hypoxia both in SKBR3 and BT474 cells, as determined by immunoblotting (Fig. 2AC), confocal immunofluorescence microscopy (Fig. 2D and E), and FACS (Fig. 2F and G). However, we found significantly decreased HER2-mediated internalization of trastuzumab at hypoxia as compared with normoxia in SKBR3 as well as BT474 cells, as visualized by confocal microscopy (Fig. 2H), and quantified by FACS (approximately 50% and 65% reduction in SKBR3 and BT474 cells, respectively; Fig. 2I). Hypoxic inhibition of trastuzumab internalization was apparent already at 30 minutes and remained up to at least 4 hours (Fig. 2J). These results suggest that hypoxia can induce resistance to T-DM1 through decreased trastuzumab/HER2 internalization.

Figure 2.

Attenuated trastuzumab internalization at tumor hypoxia mimicking conditions. Normoxic or hypoxic SKBR3 (A) and BT474 (B) cells probed for total HER2 by immunoblotting with beta-actin as loading control. C, Densitometry analysis of HER2 expressed as fraction of total beta-actin, presented as the mean ± SD from three independent experiments each performed in triplicates, NS (not significant, P > 0.05). Normoxic or hypoxic SKBR3 (D) and BT474 (E) cells were analyzed for HER2 cell surface staining by trastuzumab (TRZ) using confocal microscopy. Scale bar, (D and E) 20 μm. F, Quantitative FACS analysis of surface trastuzumab in SKBR3 cells; G, Quantitative FACS analysis of surface trastuzumab in BT474 cells. H, Normoxic or hypoxic SKBR3 and BT474 cells were analyzed for trastuzumab uptake (2 hours) by confocal microscopy. Scale bar, 10 μm. I, FACS quantification of trastuzumab uptake as in H in normoxic or hypoxic SKBR3 and BT474 cells. Data are presented as the mean ± SD from three independent experiments each performed in triplicates. J, Normoxic or hypoxic SKBR3 cells were subjected to a time course experiment to analyze trastuzumab uptake at different time points (0 hour, 30 minutes, 2 hours, and 4 hours). Scale bar, 10 μm. Images shown in D, E, H, and J, are representative of at least three independent experiments.

Figure 2.

Attenuated trastuzumab internalization at tumor hypoxia mimicking conditions. Normoxic or hypoxic SKBR3 (A) and BT474 (B) cells probed for total HER2 by immunoblotting with beta-actin as loading control. C, Densitometry analysis of HER2 expressed as fraction of total beta-actin, presented as the mean ± SD from three independent experiments each performed in triplicates, NS (not significant, P > 0.05). Normoxic or hypoxic SKBR3 (D) and BT474 (E) cells were analyzed for HER2 cell surface staining by trastuzumab (TRZ) using confocal microscopy. Scale bar, (D and E) 20 μm. F, Quantitative FACS analysis of surface trastuzumab in SKBR3 cells; G, Quantitative FACS analysis of surface trastuzumab in BT474 cells. H, Normoxic or hypoxic SKBR3 and BT474 cells were analyzed for trastuzumab uptake (2 hours) by confocal microscopy. Scale bar, 10 μm. I, FACS quantification of trastuzumab uptake as in H in normoxic or hypoxic SKBR3 and BT474 cells. Data are presented as the mean ± SD from three independent experiments each performed in triplicates. J, Normoxic or hypoxic SKBR3 cells were subjected to a time course experiment to analyze trastuzumab uptake at different time points (0 hour, 30 minutes, 2 hours, and 4 hours). Scale bar, 10 μm. Images shown in D, E, H, and J, are representative of at least three independent experiments.

Close modal

Trastuzumab uptake and T-DM1 cytotoxicity depend on CAV1 distribution

We next sought to understand the underlying mechanism of hypoxia-mediated inhibition of trastuzumab internalization and T-DM1 cytotoxicity. Our interest was focused on CAV1, i.e., a structural protein with a preference for cholesterol-rich, lipid raft membrane domains. CAV1 is generally known as a mediator of caveolar endocytosis, but has also been shown to negatively regulate extracellular ligand uptake and receptor protein internalization through plasma membrane stabilization (22, 34–37). We found no difference in total CAV1 levels between normoxia and hypoxia in both SKBR3 and BT474 cells (Fig. 3A and B). Interestingly, however, high-resolution confocal microscopy revealed that hypoxia redistributes CAV1 from intracellular vesicles to the plasma membrane in breast cancer cells (Fig. 3C and D), similarly to previous studies with HeLa and MEF cells (22). In normoxia, we found clear colocalization of internalized trastuzumab with CAV1 in cytoplasmic vesicles, whereas significantly fewer double-positive vesicles were seen in hypoxic cells (Fig. 3C and D). We next further explored the possibility that hypoxia alters CAV1 membrane microdomain distribution in breast cancer cells. Consistent with its raft association, CAV1 was found to be enriched in membrane domains with relatively low density, as shown by sucrose gradient membrane fractionation studies (Fig. 3E). However, in hypoxia, CAV1 was partly redistributed to more high-density, nonraft membrane fractions (Fig. 3E). Moreover, coimmunoprecipitation of HER2 with CAV1 appeared greater in normoxic as compared with hypoxic SKBR3 (Fig. 3F) and BT474 cells (Fig. 3G). Together, these data suggest that hypoxia redistributes CAV1 in the plasma membrane, resulting in decreased vesicular colocalization of trastuzumab and CAV1.

Figure 3.

Trastuzumab uptake and T-DM1 cytotoxicity depend on CAV1. Normoxic or hypoxic SKBR3 (A) and BT474 (B) cells were probed for total CAV1 by immunoblotting with beta-actin as loading control. Right plot: Densitometry analysis of CAV1 expressed as fraction of total beta-actin, presented as the mean ± SD from three independent experiments, each performed in triplicates, NS (not significant, P > 0.05). C, Normoxic or hypoxic SKBR3 cells were treated with trastuzumab (TRZ) for 2 hours to allow endocytosis. Cells were then stained for nuclei (blue), TRZ (green), and CAV1 (red). Yellow signal in merged images indicates colocalization. Data shown are representative of at least three independent experiments. Scale bar, 10 μm. D, Imaging data shown in C were quantified for intracellular localization of trastuzumab in CAV1+ vesicles in normoxic (N = 9) and hypoxic (N = 9) SKBR3 cells, and are presented as the mean ± SD. N = number of cells. E, Normoxic or hypoxic SKBR3 cells were subjected to subcellular sucrose density ultracentrifugation, followed by immunoblotting for CAV1 of isolated fractions. Right plot: Densitometry of CAV1 expressed as fraction of total protein in all fractions. “Heavy” and “Rafts” refer to high-density (nonraft) and low-density (cholesterol-rich) membrane fractions, respectively. Normoxic or hypoxic SKBR3 (F) and BT474 (G) cell lysates were immunoprecipitated with anti-CAV1 antibody/protein G beads and then immunoblotted for HER2 and CAV1. Normoxic or hypoxic SKBR3 or BT474 cells were used as input positive control, and protein G beads with an isotype-matched IgG as negative control. The strong bands in the lower part of the blots correspond to IgG antibody. Lower plots of (F) and (G): Densitometry of HER2 and CAV1 coimmunoprecipitated with anti-CAV1 antibody from normoxic or hypoxic SKBR3 and BT474 cells.

Figure 3.

Trastuzumab uptake and T-DM1 cytotoxicity depend on CAV1. Normoxic or hypoxic SKBR3 (A) and BT474 (B) cells were probed for total CAV1 by immunoblotting with beta-actin as loading control. Right plot: Densitometry analysis of CAV1 expressed as fraction of total beta-actin, presented as the mean ± SD from three independent experiments, each performed in triplicates, NS (not significant, P > 0.05). C, Normoxic or hypoxic SKBR3 cells were treated with trastuzumab (TRZ) for 2 hours to allow endocytosis. Cells were then stained for nuclei (blue), TRZ (green), and CAV1 (red). Yellow signal in merged images indicates colocalization. Data shown are representative of at least three independent experiments. Scale bar, 10 μm. D, Imaging data shown in C were quantified for intracellular localization of trastuzumab in CAV1+ vesicles in normoxic (N = 9) and hypoxic (N = 9) SKBR3 cells, and are presented as the mean ± SD. N = number of cells. E, Normoxic or hypoxic SKBR3 cells were subjected to subcellular sucrose density ultracentrifugation, followed by immunoblotting for CAV1 of isolated fractions. Right plot: Densitometry of CAV1 expressed as fraction of total protein in all fractions. “Heavy” and “Rafts” refer to high-density (nonraft) and low-density (cholesterol-rich) membrane fractions, respectively. Normoxic or hypoxic SKBR3 (F) and BT474 (G) cell lysates were immunoprecipitated with anti-CAV1 antibody/protein G beads and then immunoblotted for HER2 and CAV1. Normoxic or hypoxic SKBR3 or BT474 cells were used as input positive control, and protein G beads with an isotype-matched IgG as negative control. The strong bands in the lower part of the blots correspond to IgG antibody. Lower plots of (F) and (G): Densitometry of HER2 and CAV1 coimmunoprecipitated with anti-CAV1 antibody from normoxic or hypoxic SKBR3 and BT474 cells.

Close modal

Based on the above findings, we hypothesized that hypoxic resistance to T-DM1 is linked to decreased HER2/trastuzumab internalization through cellular redistribution of CAV1. To explore this possibility, we next used siRNA for transient KD of CAV1 mRNA, resulting in significant reduction of total CAV1 protein as compared with a scrambled, control siRNA sequence (Fig. 4A and B). Interestingly, we found trastuzumab uptake to be significantly reduced (approximately 20% uptake as compared with control) in SKBR3 cells displaying the most complete CAV1 KD, as assessed by quantitative image analysis from confocal microscopy (Fig. 4C and D). Notably, total HER2 protein remained intact in CAV1 KD cells, excluding that the observed effect on trastuzumab uptake was simply due to altered HER2 expression by CAV1 KD (Fig. 4A and B).

Figure 4.

CAV1 deficiency mimics the hypoxic phenotype showing reduced trastuzumab uptake and T-DM1 cytotoxicity. A, SKBR3 cells transiently transfected with scrambled control (SCR CTRL) or CAV1 siRNA (CAV1 KD) siRNA and immunoblotted for HER2 and CAV1 with beta-actin as loading control. B, Densitometry of HER2 and CAV1, expressed as fraction of total beta-actin, presented as the mean ± SD from three independent experiments. C, Quantitative analysis of internalized trastuzumab (TRZ) in SCR CTRL vs. CAV1 KD SKBR3 cells, presented as the mean ± SD from three independent experiments each performed in triplicates. D, Visualization by confocal microscopy of similar experiment as described in C. SKBR3 SCR CTRL and CAV1 KD cells were treated with TRZ to allow for internalization (2 hours). Cells were then stained for nuclei (blue), TRZ (green), and CAV1 (red). White arrow head indicates examples of cells with efficient KD of CAV1, exhibiting deficient TRZ internalization. Yellow color in merged images indicates colocalization. Images shown are representative of at least three independent experiments. Scale bar, 20 μm.

Figure 4.

CAV1 deficiency mimics the hypoxic phenotype showing reduced trastuzumab uptake and T-DM1 cytotoxicity. A, SKBR3 cells transiently transfected with scrambled control (SCR CTRL) or CAV1 siRNA (CAV1 KD) siRNA and immunoblotted for HER2 and CAV1 with beta-actin as loading control. B, Densitometry of HER2 and CAV1, expressed as fraction of total beta-actin, presented as the mean ± SD from three independent experiments. C, Quantitative analysis of internalized trastuzumab (TRZ) in SCR CTRL vs. CAV1 KD SKBR3 cells, presented as the mean ± SD from three independent experiments each performed in triplicates. D, Visualization by confocal microscopy of similar experiment as described in C. SKBR3 SCR CTRL and CAV1 KD cells were treated with TRZ to allow for internalization (2 hours). Cells were then stained for nuclei (blue), TRZ (green), and CAV1 (red). White arrow head indicates examples of cells with efficient KD of CAV1, exhibiting deficient TRZ internalization. Yellow color in merged images indicates colocalization. Images shown are representative of at least three independent experiments. Scale bar, 20 μm.

Close modal

To expand on this finding in a different setting, we employed HeLa cervical cancer cells that normally exhibit low endogenous HER2 expression (Fig 5A). Stable CAV1 KD HeLa cells were generated by lentiviral transduction, showing substantial reduction of CAV1 as compared with control cells transduced with scrambled shRNA (Fig. 5A). HER2 was introduced into HeLa cells, followed by FACS sorting (Fig. 5B) to obtain control and CAV1 KD cells expressing comparable levels of HER2, as verified by confocal microscopy and immunoblotting (Fig. 5C and D). First of all, we could show that trastuzumab internalization was inhibited by approximately 75% in hypoxic as compared with normoxic HER2+ HeLa cells, as quantified by flow cytometry (Fig. 5E, SCR CTRL). These data support our findings with SKBR3 and BT474 breast cancer cells (Fig. 2H and I), showing hypoxic downregulation of HER2/trastuzumab internalization with a comparable magnitude in HeLa cells. Also, we found a substantial reduction of trastuzumab uptake in CAV1-deficient HeLa HER2+ as compared with control HeLa HER2+ cells, both in normoxia and hypoxia (Fig. 5E, CAV1 KD), thus corroborating the results with SKBR3 cells (Fig. 4C and D). To investigate how these results translated into T-DM1 treatment effects, we performed cell viability assays, revealing that only normoxic HeLa cells with intact CAV1 expression were sensitive to T-DM1, whereas hypoxic as well as CAV1-deficient normoxic cells were relatively resistant to T-DM1 (IC50 NR; Fig. 5F and G). Together, these data suggest that CAV1 KD mimics hypoxic redistribution of CAV1, resulting in decreased HER2/trastuzumab internalization and T-DM1 cytotoxicity.

Figure 5.

HER2-overexpressing cervical cancer cells show reduced trastuzumab uptake and T-DM1 chemosensitivity when hypoxic or CAV1-deficient. A, HeLa cervical cancer cells stably transduced with shRNA targeting CAV1 mRNA (CAV1 KD) or with a scrambled shRNA sequence (SCR CTRL) were immunoblotted for HER2 and CAV1 with β-actin as loading control. B, Representative FACS scatter plots showing HER2+ (green) and HER2 (red) CAV1 KD and SCR CTRL HeLa cells transfected with HER2 DNA plasmid. Visualization of FACS-sorted HER2+ HeLa cell lines by confocal immunofluorescence microscopy (C) and immunoblotting (D) indicates comparable HER2 levels in CAV1 KD and SCR CTRL cells. E, FACS quantification of trastuzumab uptake (2 hours) in normoxic or hypoxic HER2-SCR CTRL and HER2-CAV1 KD HeLa cells. Data are presented as the mean ± SD from three independent experiments, each performed in triplicates. Dose–response curve showing sensitivity of normoxic or hypoxic HER2-CAV1 KD (F) and HER2-SCR CTRL (G) cells treated with T-DM1 for 72 hours. Cell viability was measured by MTS assay and expressed as percent cell viability of control. NR, not reached.

Figure 5.

HER2-overexpressing cervical cancer cells show reduced trastuzumab uptake and T-DM1 chemosensitivity when hypoxic or CAV1-deficient. A, HeLa cervical cancer cells stably transduced with shRNA targeting CAV1 mRNA (CAV1 KD) or with a scrambled shRNA sequence (SCR CTRL) were immunoblotted for HER2 and CAV1 with β-actin as loading control. B, Representative FACS scatter plots showing HER2+ (green) and HER2 (red) CAV1 KD and SCR CTRL HeLa cells transfected with HER2 DNA plasmid. Visualization of FACS-sorted HER2+ HeLa cell lines by confocal immunofluorescence microscopy (C) and immunoblotting (D) indicates comparable HER2 levels in CAV1 KD and SCR CTRL cells. E, FACS quantification of trastuzumab uptake (2 hours) in normoxic or hypoxic HER2-SCR CTRL and HER2-CAV1 KD HeLa cells. Data are presented as the mean ± SD from three independent experiments, each performed in triplicates. Dose–response curve showing sensitivity of normoxic or hypoxic HER2-CAV1 KD (F) and HER2-SCR CTRL (G) cells treated with T-DM1 for 72 hours. Cell viability was measured by MTS assay and expressed as percent cell viability of control. NR, not reached.

Close modal

To further substantiate that CAV1-dependent uptake is altered in hypoxic conditions, we performed cell viability assays with Abraxane, i.e., a paclitaxel-albumin nanoparticle drug approved in the treatment of breast cancer that is known to enter cells via a CAV1-associated pathway (38). Abraxane was highly toxic to normoxic SKBR3 cells (IC50 7.0 nmol/L), whereas hypoxic cells were relatively resistant (IC50 NR; Supplementary Fig. S4). These results further support the notion that hypoxic conditions can attenuate the efficacy of macromolecular drugs that enter target cells through CAV1-mediated pathways.

Hypoxic redistribution of phosphorylated CAV1 and T-DM1 resistance

Previous studies have implicated a role for CAV1 tyrosine phosphorylation in protein internalization (39–43). More specifically, del Pozo and colleagues (42) showed that redistribution of phosphorylated CAV1 (pCAV1) from focal adhesions to endosomes is associated with increased endocytosis. Moreover, cellular stress (e.g., high osmolarity) was shown to increase CAV1 phosphorylation on tyrosine 14 and the localization of pCAV1 to the plasma membrane/focal adhesions (44). We next explored the possibility that inhibition of trastuzumab internalization and T-DM1 cytotoxicity by hypoxic stress is related to altered CAV1 phosphorylation and/or pCAV1 subcellular distribution. We did not observe any difference in total pCAV1 between normoxic and hypoxic conditions in SKBR3 cells (Fig. 6A); however, membrane fractionation studies suggested a relative depletion of pCAV1 in membrane raft regions of hypoxic as compared with normoxic cell lysates (fractions 7 and 8; Fig. 6B). Moreover, confocal microscopy studies demonstrated clear colocalization of internalized trastuzumab and pCAV1 in vesicular structures of normoxic cells, whereas significantly fewer double-positive vesicles were seen in hypoxic cells (Fig. 6CE).

Figure 6.

Hypoxic redistribution of pCAV1 and reduced trastuzumab uptake and T-DM1 cytotoxicity by overexpression of a phosphorylation-deficient CAV1 mutant. A, Normoxic or hypoxic SKBR3 cells were probed for pCAV1 by immunoblotting with β-actin as loading control. Data shown are representative of two independent experiments. B, Normoxic or hypoxic SKBR3 cells were subjected to subcellular sucrose density ultracentrifugation, followed by immunoblotting for pCAV1 in isolated membrane density fractions. Right plot: Densitometry of pCAV1, expressed as fraction of total protein in all fractions. “Heavy” and “Rafts” refer to high-density (nonraft) and low-density (cholesterol-rich) membrane fractions, respectively. Data shown are representative of three independent experiments. Normoxic (C) or hypoxic (D) SKBR3 cells were treated with trastuzumab to allow endocytosis (2 hours). Cells were then stained for nuclei (blue), trastuzumab (green), and pCAV1 (red). Yellow in merged images indicates colocalization. Scale bar, 10 μm. E, Imaging data shown in C and D were quantified for intracellular localization of trastuzumab in pCAV1+ vesicles in normoxic (N = 13) and hypoxic (N = 12) SKBR3 cells, and presented as the mean ± SD. N = number of cells. SKBR3 cells transfected with phosphomimicking CAV1 (Y14D-CAV1-RFP; F) or phosphorylation-deficient, dominant-negative CAV1-mutant (Y14F-CAV1-RFP; G) were treated with trastuzumab to allow endocytosis (2 hours). Cells were then stained for nuclei (blue) and trastuzumab (green) for confocal immunofluorescence analysis. Yellow in merged images indicates colocalization. Scale bar, 20 μm. H, Imaging data shown in F and G were quantified for intracellular localization of trastuzumab in Y14D-CAV1-RFP (N = 50) and Y14F-CAV1-RFP (N = 58) SKBR3 cells. N = number of cells. I, FACS quantification of trastuzumab uptake in SKBR3 cells transfected with wild-type CAV1 (WT), phosphomimicking CAV1 (Y14D-CAV1-RFP), or phosphorylation-deficient, dominant-negative CAV1 mutant (Y14F-CAV1-RFP). Data are presented as the mean ± SD from three independent experiments, each performed in triplicates. J, Dose–response curve showing sensitivity of SKBR3 cells treated with T-DM1 for 72 hours. Cell viability was measured by MTS assay and expressed as percent cell viability of control. NR, not reached.

Figure 6.

Hypoxic redistribution of pCAV1 and reduced trastuzumab uptake and T-DM1 cytotoxicity by overexpression of a phosphorylation-deficient CAV1 mutant. A, Normoxic or hypoxic SKBR3 cells were probed for pCAV1 by immunoblotting with β-actin as loading control. Data shown are representative of two independent experiments. B, Normoxic or hypoxic SKBR3 cells were subjected to subcellular sucrose density ultracentrifugation, followed by immunoblotting for pCAV1 in isolated membrane density fractions. Right plot: Densitometry of pCAV1, expressed as fraction of total protein in all fractions. “Heavy” and “Rafts” refer to high-density (nonraft) and low-density (cholesterol-rich) membrane fractions, respectively. Data shown are representative of three independent experiments. Normoxic (C) or hypoxic (D) SKBR3 cells were treated with trastuzumab to allow endocytosis (2 hours). Cells were then stained for nuclei (blue), trastuzumab (green), and pCAV1 (red). Yellow in merged images indicates colocalization. Scale bar, 10 μm. E, Imaging data shown in C and D were quantified for intracellular localization of trastuzumab in pCAV1+ vesicles in normoxic (N = 13) and hypoxic (N = 12) SKBR3 cells, and presented as the mean ± SD. N = number of cells. SKBR3 cells transfected with phosphomimicking CAV1 (Y14D-CAV1-RFP; F) or phosphorylation-deficient, dominant-negative CAV1-mutant (Y14F-CAV1-RFP; G) were treated with trastuzumab to allow endocytosis (2 hours). Cells were then stained for nuclei (blue) and trastuzumab (green) for confocal immunofluorescence analysis. Yellow in merged images indicates colocalization. Scale bar, 20 μm. H, Imaging data shown in F and G were quantified for intracellular localization of trastuzumab in Y14D-CAV1-RFP (N = 50) and Y14F-CAV1-RFP (N = 58) SKBR3 cells. N = number of cells. I, FACS quantification of trastuzumab uptake in SKBR3 cells transfected with wild-type CAV1 (WT), phosphomimicking CAV1 (Y14D-CAV1-RFP), or phosphorylation-deficient, dominant-negative CAV1 mutant (Y14F-CAV1-RFP). Data are presented as the mean ± SD from three independent experiments, each performed in triplicates. J, Dose–response curve showing sensitivity of SKBR3 cells treated with T-DM1 for 72 hours. Cell viability was measured by MTS assay and expressed as percent cell viability of control. NR, not reached.

Close modal

Together, these data are consistent with analyses of total CAV1 (Fig. 4) and suggest a specific role of the phosphorylated fraction of CAV1 in the regulation of trastuzumab uptake. To test this idea more directly, we next generated SKBR3 cells expressing either wild-type CAV1-RFP (WT-CAV1-RFP, control), Y14D-CAV1-RFP, i.e., a CAV1 tyrosine phosphorylation mimic, or phosphorylation-deficient Y14F-CAV1-RFP that exhibits dominant-negative activity. Consistent with studies by Zimnicka and colleagues in endothelial cells (43), we could show that SKBR3 cells expressing phosphomimicking CAV1 (Y14D-CAV1-RFP) as compared with phosphorylation-deficient CAV1-mutant cells (Y14F-CAV1-RFP) display a higher number of vesicular structures (Fig. 6F and G). More importantly, trastuzumab uptake was significantly decreased in Y14F-CAV1-RFP as compared with WT-CAV1-RFP and Y14D-CAV1-RFP SKBR3 cell transfectants, as quantified by imaging analysis (Fig. 6H) as well as by FACS (approximately 30% uptake in Y14F-CAV1-RFP as compared with WT-CAV1-RFP; Fig. 6I). In cell viability studies using the same set of CAV1 SKBR3 transfectants, IC50 was not reached in Y14F-CAV1-RFP, whereas in WT-CAV1-RFP and Y14D-CAV1-RFP cells, IC50 was reached at 2.0 nmol/L (Fig. 6J). From these data, we conclude that pCAV1 has a role in regulating trastuzumab internalization and T-DM1 cytotoxicity, and that this function is perturbed by hypoxic conditions.

Expression of cytoplasmic CAV1 and HER2 in human breast cancer

The above data suggested a dependency on CAV1 vesicular localization for the function of the internalizing HER2 fraction, which constitutes a small fraction of total surface HER2. Notably, in cell studies, CAV1 did not seem to regulate total HER2 expression (Fig. 4) and vice versa (Fig. 5). To investigate how these observations are reflected in human tumors and to further explore if there is any direct association between HER2 amplification status and CAV1 protein expression, we employed a well-annotated, population-based cohort of patients with breast cancer (Supplementary Fig. S5; ref. 29). Cytoplasmic CAV1 expression was denoted in at least 20% of all stained invasive tumor cells in 274 of 635 patient samples (approximately 43% of total). Among these, 231 tumors displayed weak, 39 moderate, and 4 strong CAV1 expression (Supplementary Fig. S6). A higher level of CAV1 was observed in adjacent carcinoma in situ (CIS) cells compared with invasive tumor cells. In benign appearing ducts as well as CIS, the CAV1 level was higher in the myoepithelial compared with the luminal cells (Supplementary Fig. S7). Due to the limited number of tumors with strong CAV1 expression, these tumors were grouped with tumors of moderate intensity for further analyses. We found no significant associations between CAV1 expression and patient age at inclusion, invasive tumor size, or axillary lymph node positivity (Supplementary Table S1). CAV1 expression showed a significant inverse association with estrogen receptor (ER) and progesterone receptor status. Importantly, however, there was no association of CAV1 expression with HER2 amplification status (Supplementary Table S1). We conclude that, in line with the in vitro data, there is no apparent coregulatory association between HER2 and cytoplasmic CAV1 expression.

Since the advent of HER2 targeted therapies, the clinical outcome for patients with breast cancer with HER2+ tumors has improved dramatically. Still, there is evidence of significant development of resistance to trastuzumab as well as to T-DM1, posing a clinical challenge that may be resolved by an increased understanding of HER2 function in the context of the tumor microenvironment. Here, we demonstrate that hypoxia, i.e., a specific and universal feature of aggressive tumors, confers resistance to T-DM1. It is suggested that hypoxia-induced T-DM1 resistance is the result of deficient trastuzumab/HER2 internalization due to redistribution of pCAV1 from vesicular membrane raft domains to the plasma membrane. These findings reveal a novel link between tumor hypoxic stress conditions, HER2 trafficking, and T-DM1 chemosensitivity with potential implications for improved therapeutic strategies and response prediction.

Based on the present findings together with previous studies (22), different categories of cell surface receptors in relation to hypoxia and CAV1 may be proposed. These include proteins that are independent on CAV1 for their endocytosis and that may escape the negative regulation by CAV1 redistribution in hypoxia (e.g., CAIX); proteins that are independent on CAV1 in normoxia but are negatively regulated by CAV1 membrane stabilization in hypoxia; and proteins, including HER2, that are dependent on CAV1 for their endocytosis and negatively regulated by hypoxic redistribution of CAV1. This implies that the inhibitory effect of hypoxia on receptor protein internalization may in some cases be alleviated by the loss of CAV1, whereas in others the hypoxic situation would instead be mimicked by deficient CAV1 or CAV1 phosphorylation, as shown for HER2 in the present study. Our data thus point at a scenario where hypoxia perturbs CAV1 function as an important mediator of HER2 tumor antigen internalization and ADC delivery. We show that CAV1 moves from low-density (raft) to more high-density membrane regions, which may be the result of, for example, hypoxia-induced remodeling of membrane lipid composition or altered kinase activation, perhaps most importantly Src and p38 MAP kinases that previously have been associated with stress and regulation of caveolae function (44). Another possible mechanism of how hypoxia redistributes CAV1 is by increased expression or activation of a protein interaction partner that preferentially localizes to high-density plasma membrane regions. Clearly, a key remaining question for future studies is to understand exactly how hypoxia regulates CAV1 function.

Our data illustrate the limitations of studies that extrapolate genomic data or overall protein expression to biological function, and highlight the importance of adding spatial information on protein distribution. Notably, the current status of patient selection for T-DM1 treatment does not take into account the subcellular distribution of HER2, and therefore may overestimate accessible HER2 that can engage in T-DM1 internalization. Accordingly, previous PET imaging studies demonstrated a lack of correlation between HER2 expression and tumor uptake of trastuzumab (45, 46), suggesting that assessment of HER2 alone is insufficient to predict how patients respond to T-DM1. The present study provides a potential mechanistic explanation to this notion and should motivate future studies that explore how markers of tumor hypoxia and CAV1 phosphorylation may serve to better predict therapeutic vulnerability of HER2+ tumors to T-DM1 treatment.

Of particular relevance in breast cancer, CAV1 may directly regulate HER2 and ER signaling activity (47–49). Indeed, previous studies have demonstrated that HER2 is localized to cholesterol-rich membrane raft regions where it may colocalize with GM1 (50) as well as CAV1 (51). Recent studies by Pereira and colleagues (52) suggested an inverse relationship between HER2 and CAV1 protein expression in a large collection of cancer cell lines and in examples of human gastric tumor specimens. In the present study, we were, however, unable to find a significant association between CAV1 expression and HER2 status in vitro as well as in a large, population-based breast cancer cohort. As the cohort was established prior to the introduction of T-DM1 treatment, we were unable to perform correlative analyses between CAV1 expression and response to T-DM1 treatment. Caveolae-mediated endocytosis is one of the major clathrin-independent raft-dependent endocytic routes (53), and studies on its role in trastuzumab uptake and T-DM1 cytotoxicity have generated conflicting data. Chung and colleagues (26) suggested that CAV1 promotes chemosensitivity to T-DM1, whereas others, on the contrary, reported that the acquisition of TDM1 resistance is due to deficient cleavage of T-DM1 in CAV1-associated endosomes (28). In favor of a role of CAV1 in promoting T-DM1 sensitivity, increased CAV1 expression by metformin was shown to induce T-DM1 internalization and subsequent cytotoxicity (27). Our data strongly support the concept of a dependence on vesicular, raft-associated CAV1 for efficient T-DM1 cytotoxic activity. In addition, we show a specific role for pCAV1 in trastuzumab internalization and T-DM1 sensitivity that, however, is disrupted in the context of tumor hypoxic conditions. According to this scenario, the most obvious strategy would be to alleviate the hypoxic situation by, for example, tumor vessel normalization or actions that normalize CAV1 localization to allow more efficient targeting also of hypoxic tumor regions and potentially other niches distinguished by CAV1 clustering at the cell periphery. Interestingly, in models of gastric, bladder, and breast cancer, increased HER2 cell surface availability and improved trastuzumab therapy by disrupting CAV1-mediated HER2 internalization using the cholesterol lowering drug lovastatin were demonstrated (52). In the case of T-DM1, however, one would like to achieve the opposite effect, i.e., increased membrane cholesterol loading and CAV1-mediated HER2 internalization.

In summary, we show that hypoxic conditions induce resistance to targeted treatment with T-DM1 in HER2+ breast cancer cells and HER2-overexpressing cervical cancer cells by perturbing the localization and function of CAV1. Our findings may have direct implications for improved targeting and response prediction of HER2-expressing tumors.

O.M. Saad is Senior Scientist at Genentech/Roche and has an ownership interest (including patents) in Roche Stock. O. Gluz is an advisory board member for Roche and is employed with Daiichi. S. Borgquist is clinical advisor at Pfizer, reports receiving other commercial research support from Roche, and has honoraria from the speakers' bureau of Pfizer. H. Jernström has an ownership interest (including patents) in Pfizer stocks. No potential conflicts of interest were disclosed by the other authors.

Conception and design: V. Indira Chandran, M. Belting

Development of methodology: V. Indira Chandran, M. Cerezo-Magaña, I.R. Nabi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): V. Indira Chandran, B. Nodin, N. Koppada, O.M. Saad, S. Borgquist, H. Jernström, M. Belting

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V. Indira Chandran, M. Barbachowska, M. Cerezo-Magaña, N. Koppada, O.M. Saad, O. Gluz, H. Jernström, M. Belting

Writing, review, and/or revision of the manuscript: V. Indira Chandran, M. Cerezo-Magaña, O. Gluz, K. Isaksson, S. Borgquist, K. Jirström, I.R. Nabi, H. Jernström, M. Belting

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): V. Indira Chandran, A.-S. Månsson, M. Barbachowska, B. Nodin, K. Jirström, H. Jernström

Study supervision: V. Indira Chandran, H. Jernström, M. Belting

Other (Second reader for TMA scoring and author of selected TMA images): M. Barbachowska

Other (some of the Caveolin constructs were provided): B. Joshi

We thank Maria C. Johansson for excellent technical assistance and all the patients who contributed to this study.

This study was funded by grants (to M. Belting) from the Swedish Cancer Society (CAN 2017/664); the Swedish Research Council (2018-02562); the Mrs. Berta Kamprad Foundation; the Skåne University Hospital donation funds; the Governmental funding of clinical research within the national health services, ALF; and a donation by Viveca Jeppsson.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Slamon
DJ
,
Clark
GM
,
Wong
SG
,
Levin
WJ
,
Ullrich
A
,
McGuire
WL
. 
Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene
.
Science
1987
;
235
:
177
82
.
2.
Slamon
DJ
,
Godolphin
W
,
Jones
LA
,
Holt
JA
,
Wong
SG
,
Keith
DE
, et al
Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer
.
Science
1989
;
244
:
707
12
.
3.
Press
MF
,
Bernstein
L
,
Thomas
PA
,
Meisner
LF
,
Zhou
JY
,
Ma
Y
, et al
HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas
.
J Clin Oncol
1997
;
15
:
2894
904
.
4.
Leonard
DS
,
Hill
AD
,
Kelly
L
,
Dijkstra
B
,
McDermott
E
,
O'Higgins
NJ
. 
Anti-human epidermal growth factor receptor 2 monoclonal antibody therapy for breast cancer
.
Brit J Surg
2002
;
89
:
262
71
.
5.
Moasser
MM
. 
Targeting the function of the HER2 oncogene in human cancer therapeutics
.
Oncogene
2007
;
26
:
6577
92
.
6.
Phillips
GDL
,
Li
G
,
Dugger
DL
,
Crocker
LM
,
Parsons
KL
,
Mai
E
, et al
Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate
.
Cancer Res
2008
;
68
:
9280
90
.
7.
Verma
S
,
Miles
D
,
Gianni
L
,
Krop
IE
,
Welslau
M
,
Baselga
J
, et al
Trastuzumab emtansine for HER2-positive advanced breast cancer
.
N Engl J Med
2012
;
367
:
1783
91
.
8.
von Minckwitz
G
,
Huang
CS
,
Mano
MS
,
Loibl
S
,
Mamounas
EP
,
Untch
M
, et al
Trastuzumab emtansine for residual invasive HER2-positive breast cancer
.
N Engl J Med
2019
;
380
:
617
28
.
9.
Li
BT
,
Shen
R
,
Buonocore
D
,
Olah
ZT
,
Ni
A
,
Ginsberg
MS
, et al
Ado-trastuzumab emtansine for patients with HER2-mutant lung cancers: results from a phase II basket trial
.
J Clin Oncol
2018
;
36
:
2532
7
.
10.
Ríos-Luci
C
,
García-Alonso
S
,
Díaz-Rodríguez
E
,
Nadal-Serrano
M
,
Arribas
J
,
Ocaña
A
, et al
Resistance to the antibody–drug conjugate T-DM1 is based in a reduction in lysosomal proteolytic activity
.
Cancer Res
2017
;
77
:
4639
51
.
11.
Saatci
O
,
Borgoni
S
,
Akbulut
Ö
,
Durmuş
S
,
Raza
U
,
Eyüpoğlu
E
, et al
Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2/xL in HER2-positive breast cancer
.
Oncogene
2018
;
37
:
2251
69
.
12.
Sabbaghi
M
,
Gil-Gómez
G
,
Guardia
C
,
Servitja
S
,
Arpí
O
,
García-Alonso
S
, et al
Defective cyclin B1 induction in trastuzumab-emtansine (T-DM1) acquired resistance in HER2-positive breast cancer
.
Clin Cancer Res
2017
;
23
:
7006
19
.
13.
Loganzo
F
,
Tan
X
,
Sung
M
,
Jin
G
,
Myers
JS
,
Melamud
E
, et al
Tumor cells chronically treated with a trastuzumab-maytansinoid antibody-drug conjugate develop varied resistance mechanisms but respond to alternate treatments
.
Mol Cancer Ther
2015
;
14
:
952
63
.
14.
Li
G
,
Guo
J
,
Shen
BQ
,
Yadav
DB
,
Sliwkowski
MX
,
Crocker
LM
, et al
Mechanisms of acquired resistance to trastuzumab emtansine in breast cancer cells
.
Mol Cancer Ther
2018
;
17
:
1441
53
.
15.
Liu
D
,
Yang
Z
,
Wang
T
,
Yang
Z
,
Chen
H
,
Hu
Y
, et al
beta2-AR signaling controls trastuzumab resistance-dependent pathway
.
Oncogene
2016
;
35
:
47
58
.
16.
Valabrega
G
,
Montemurro
F
,
Sarotto
I
,
Petrelli
A
,
Rubini
P
,
Tacchetti
C
, et al
TGFalpha expression impairs trastuzumab-induced HER2 downregulation
.
Oncogene
2005
;
24
:
3002
10
.
17.
Brown
JM
,
Wilson
WR
. 
Exploiting tumor hypoxia in cancer treatment
.
Nat Rev Cancer
2004
;
4
:
437
47
.
18.
Tredan
O
,
Galmarini
CM
,
Patel
K
,
Tannock
IF
. 
Drug resistance and the solid tumor microenvironment
.
J Natl Cancer Inst
2007
;
99
:
1441
54
.
19.
Hockel
M
,
Vaupel
P
. 
Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects
.
J Natl Cancer Inst
2001
;
93
:
266
76
.
20.
Wang
Y
,
Roche
O
,
Yan
MS
,
Finak
G
,
Evans
AJ
,
Metcalf
JL
, et al
Regulation of endocytosis via the oxygen-sensing pathway
.
Nat Med
2009
;
15
:
319
24
.
21.
Wang
Y
,
Roche
O
,
Xu
C
,
Moriyama
EH
,
Heir
P
,
Chung
J
, et al
Hypoxia promotes ligand-independent EGF receptor signaling via hypoxia-inducible factor-mediated upregulation of caveolin-1
.
Proc Natl Acad Sci U S A
2012
;
109
:
4892
7
.
22.
Bourseau-Guilmain
E
,
Menard
JA
,
Lindqvist
E
,
Indira Chandran
V
,
Christianson
HC
,
Cerezo Magaña
M
, et al
Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells
.
Nat Commun
2016
;
7
:
11371
.
23.
Nagy
P
,
Vereb
G
,
Sebestyén
Z
,
Horváth
G
,
Lockett
SJ
,
Damjanovich
S
, et al
Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2
.
J Cell Sci
2002
;
115
:
4251
62
.
24.
Nagy
P
,
Claus
J
,
Jovin
TM
,
Arndt-Jovin
DJ
. 
Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis
.
Proc Natl Acad Sci U S A
2010
;
107
:
16524
9
.
25.
Pust
S
,
Klokk
TI
,
Musa
N
,
Jenstad
M
,
Risberg
B
,
Erikstein
B
, et al
Flotillins as regulators of ErbB2 levels in breast cancer
.
Oncogene
2013
;
32
:
3443
51
.
26.
Chung
YC
,
Kuo
JF
,
Wei
WC
,
Chang
KJ
,
Chao
WT
. 
Caveolin-1 dependent endocytosis enhances the chemosensitivity of HER-2 positive breast cancer cells to trastuzumab emtansine (T-DM1)
.
PloS one
2015
;
10
:
e0133072
.
27.
Chung
YC
,
Chang
CM
,
Wei
WC
,
Chang
TW
,
Chang
KJ
,
Chao
WT
. 
Metformin-induced caveolin-1 expression promotes T-DM1 drug efficacy in breast cancer cells
.
Sci Rep
2018
;
8
:
3930
.
28.
Sung
M
,
Tan
X
,
Lu
B
,
Golas
J
,
Hosselet
C
,
Wang
F
, et al
Caveolae-mediated endocytosis as a novel mechanism of resistance to trastuzumab emtansine (T-DM1)
.
Mol Cancer Ther
2018
;
17
:
243
53
.
29.
Simonsson
M
,
Björner
S
,
Markkula
A
,
Nodin
B
,
Jirström
K
,
Rose
C
, et al
The prognostic impact of COX-2 expression in breast cancer depends on oral contraceptive history, preoperative NSAID use, and tumor size
.
Int J Cancer
2017
;
140
:
163
75
.
30.
Joshi
B
,
Bastiani
M
,
Strugnell
SS
,
Boscher
C
,
Parton
RG
,
Nabi
IR
. 
Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation
.
J Cell Biol
2012
;
199
:
425
35
.
31.
Swaney
JS
,
Patel
HH
,
Yokoyama
U
,
Head
BP
,
Roth
DM
,
Insel
PA
. 
Focal adhesions in (myo)fibroblasts scaffold adenylyl cyclase with phosphorylated caveolin
.
J Biol Chem
2006
;
281
:
17173
9
.
32.
Junttila
TT
,
Li
G
,
Parsons
K
,
Phillips
GL
,
Sliwkowski
MX
. 
Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer
.
Breast Cancer Res Treat
2011
;
128
:
347
56
.
33.
Erickson
HK
,
Park
PU
,
Widdison
WC
,
Kovtun
YV
,
Garrett
LM
,
Hoffman
K
, et al
Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing
.
Cancer Res
2006
;
66
:
4426
33
.
34.
Lajoie
P
,
Nabi
IR
. 
Lipid rafts, caveolae, and their endocytosis
.
Int Rev Cell Mol Biol
2010
;
282
:
135
63
.
35.
Parton
RG
,
del Pozo
MA
. 
Caveolae as plasma membrane sensors, protectors and organizers
.
Nat Rev Mol Cell Biol
2013
;
14
:
98
112
.
36.
Shvets
E
,
Bitsikas
V
,
Howard
G
,
Hansen
CG
,
Nichols
BJ
. 
Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids
.
Nat Commun
2015
;
6
:
6867
.
37.
Svensson
KJ
,
Christianson
HC
,
Wittrup
A
,
Bourseau-Guilmain
E
,
Lindqvist
E
,
Svensson
LM
, et al
Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1
.
J Biol Chem
2013
;
288
:
17713
24
.
38.
Chatterjee
M
,
Ben-Josef
E
,
Robb
R
,
Vedaie
M
,
Seum
S
,
Thirumoorthy
K
, et al
Caveolae-mediated endocytosis is critical for albumin cellular uptake and response to albumin-bound chemotherapy
.
Cancer Res
2017
;
77
:
5925
37
.
39.
Parton
RG
,
Joggerst
B
,
Simons
K
. 
Regulated internalization of caveolae
.
J Cell Biol
1994
;
127
:
1199
215
.
40.
Aoki
T
,
Nomura
R
,
Fujimoto
T
. 
Tyrosine phosphorylation of caveolin-1 in the endothelium
.
Exp Cell Res
1999
;
253
:
629
36
.
41.
Pelkmans
L
,
Puntener
D
,
Helenius
A
. 
Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae
.
Science
2002
;
296
:
535
9
.
42.
del Pozo
MA
,
Balasubramanian
N
,
Alderson
NB
,
Kiosses
WB
,
Grande-García
A
,
Anderson
RG
, et al
Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization
.
Nat Cell Biol
2005
;
7
:
901
8
.
43.
Zimnicka
AM
,
Husain
YS
,
Shajahan
AN
,
Sverdlov
M
,
Chaga
O
,
Chen
Z
, et al
Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
.
Mol Biol Cell
2016
;
27
:
2090
106
.
44.
Volonte
D
,
Galbiati
F
,
Pestell
RG
,
Lisanti
MP
. 
Cellular stress induces the tyrosine phosphorylation of caveolin-1 (Tyr(14)) via activation of p38 mitogen-activated protein kinase and c-Src kinase. Evidence for caveolae, the actin cytoskeleton, and focal adhesions as mechanical sensors of osmotic stress
.
J Biol Chem
2001
;
276
:
8094
103
.
45.
Ulaner
GA
,
Hyman
DM
,
Ross
DS
,
Corben
A
,
Chandarlapaty
S
,
Goldfarb
S
, et al
Detection of HER2-positive metastases in patients with HER2-negative primary breast cancer using 89Zr-trastuzumab PET/CT
.
J Nucl Med
2016
;
57
:
1523
8
.
46.
Gebhart
G
,
Lamberts
LE
,
Wimana
Z
,
Garcia
C
,
Emonts
P
,
Ameye
L
, et al
Molecular imaging as a tool to investigate heterogeneity of advanced HER2-positive breast cancer and to predict patient outcome under trastuzumab emtansine (T-DM1): the ZEPHIR trial
.
Ann Oncol
2016
;
27
:
619
24
.
47.
Engelman
JA
,
Lee
RJ
,
Karnezis
A
,
Bearss
DJ
,
Webster
M
,
Siegel
P
, et al
Reciprocal regulation of neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Implications for human breast cancer
.
J Biol Chem
1998
;
273
:
20448
55
.
48.
Schlegel
A
,
Wang
C
,
Katzenellenbogen
BS
,
Pestell
RG
,
Lisanti
MP
. 
Caveolin-1 potentiates estrogen receptor alpha (ERalpha) signaling. caveolin-1 drives ligand-independent nuclear translocation and activation of ERalpha
.
J Biol Chem
1999
;
274
:
33551
6
.
49.
Duffy
MJ
,
Harbeck
N
,
Nap
M
,
Molina
R
,
Nicolini
A
,
Senkus
E
, et al
Clinical use of biomarkers in breast cancer: Updated guidelines from the European Group on Tumor Markers (EGTM)
.
Eur J Cancer
2017
;
75
:
284
98
.
50.
Hommelgaard
AM
,
Lerdrup
M
,
van Deurs
B
. 
Association with membrane protrusions makes ErbB2 an internalization-resistant receptor
.
Mol Biol Cell
2004
;
15
:
1557
67
.
51.
Raina
D
,
Uchida
Y
,
Kharbanda
A
,
Rajabi
H
,
Panchamoorthy
G
,
Jin
C
, et al
Targeting the MUC1-C oncoprotein downregulates HER2 activation and abrogates trastuzumab resistance in breast cancer cells
.
Oncogene
2014
;
33
:
3422
31
.
52.
Pereira
PMR
,
Sharma
SK
,
Carter
LM
,
Edwards
KJ
,
Pourat
J
,
Ragupathi
A
, et al
Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
.
Nat Commun
2018
;
9
:
5137
.
53.
Nabi
IR
,
Le
PU
. 
Caveolae/raft-dependent endocytosis
.
J Cell Biol
2003
;
161
:
673
7
.

Supplementary data