Caveolin-1 temporal modulation enhances antibody drug efficacy in heterogeneous gastric cancer

Abstract

Resistance mechanisms and heterogeneity in HER2-positive gastric cancers (GC) limit Trastuzumab benefit in 32% of patients, and other targeted therapies have failed in clinical trials. Using patient samples, patient-derived xenografts (PDXs), partially humanized biological models, and HER2-targeted imaging technologies we demonstrate the role of caveolin-1 (CAV1) as a complementary biomarker in GC selection for Trastuzumab therapy. In retrospective analyses of samples from patients enrolled on Trastuzumab trials, the CAV1-high profile associates with low membrane HER2 density and low patient survival. We show a negative correlation between CAV1 tumoral protein levels - a major protein of cholesterol-rich membrane domains - and Trastuzumab-drug conjugate TDM1 tumor uptake. Finally, CAV1 depletion using knockdown or pharmacologic approaches (statins) increases antibody drug efficacy in tumors with incomplete HER2 membranous reactivity. In support of these findings, background statin use in patients associates with enhanced antibody efficacy. Together, this work provides preclinical justification and clinical evidence that require prospective investigation of antibody drugs combined with statins to delay drug resistance in tumors.

Fig. 1. HER2 membrane levels and Trastuzumab efficacy depend on CAV1 protein levels.

a Immunohistochemical (IHC) detection and scoring intensity of CAV1, immunofluorescence (IF) staining of HER2 (green color) and CAV1 (red color) in HER2-expressing gastric tumor tissues. CAV1 reactivity at the cell membrane of tumor cells was considered for IHC scoring; IHC 0/1 + : CAV1-low (patient #14 and patients #3–5). IHC 2 + /3 + : CAV1-high (patient #1 and patient #2). The graphs plot protein fluorescence intensity per unit area, calculated by quantifying IF images (mean ± S.E.M, n = 3). Scale bar, 50 μm. HER2 membrane levels are classified as high versus low based on quantification of IF staining shown in Supplementary Fig. 5. Patient 1 to Patient 33 are IDs for all HER2⁺ gastric tumor tissues analyzed in the study (Supplementary Fig. 4). b ⁸⁹Zr-labeled Trastuzumab (1 μCi, 0.25 μg) binding and internalization in NCIN87 GC cells wild-type (WT) and AGS, KATOIII, SNU1 GC sublines stably expressing HER2 (LV-HER2). c Kaplan–Meier analyses of CAV1 expression and GC disease outcome in patients treated with Trastuzumab. Patients with HER2⁺/CAV1^HIGH (blue color, n = 12 patients) phenotype have a worse survival than HER2⁺/CAV1^LOW (red, n = 34 patients). Log rank; p < 1 × 10⁻⁴. Source data are provided as a Source Data file.

Fig. 2. CAV1 depletion enhances TDM1-tumor binding.

a [⁸⁹Zr]Zr-DFO-TDM1 uptake in HER2-expressing gastric PDXs containing varying levels of CAV1. NSG mice bearing subcutaneous PDXs were intravenously administered with [⁸⁹Zr]Zr-DFO-TDM1 (6.66–7.4 Mbq, 45–50 μg protein) and biodistribution performed at 48 h p.i. of ⁸⁹Zr-labeled antibody. PDX IDs in this figure match patient IDs shown in Fig. 1. Points, n = 5 mice per group, mean ± S.E.M. %ID/g, percentage of injected dose per gram. b–d Athymic nude mice bearing s.c. NCIN87 shRNA NTC, shRNA 486, or shRNA 479 xenografts were orally administered with 10 mg/mL of Dox (ON DOX) or PBS (OFF DOX) for 11 days. On day 11, mice were intravenously administered with [⁸⁹Zr]Zr-DFO-TDM1 (6.66–7.4 Mbq, 45–50 μg protein). PET images (c) were recorded at 24, 48, and 72 h p.i. [⁸⁹Zr]Zr-DFO-TDM1. The percentage of injected dose per gram (%ID/g) of TDM1 in tumors (d) was calculated by quantifying regions of interest (ROIs) in the PET images. *P < 0.05 based on a Student’s t test, n = 3. Source data are provided as a Source Data file.

Fig. 3. Statins enhance TDM1 binding and internalization.

a Confocal images of immunofluorescence of TDM1 in the presence or absence of lovastatin. Scale bars: 20 μm. b TDM1 internalization and recycling in NCIN87 GC cells wild-type (WT) and AGS, KATOIII, SNU1 GC sublines stably expressing HER2 (LV-HER2) in the presence and absence of lovastatin. The pHrodo-TDM1 fluorescent signal was normalized to the number of viable cells (*P < 0.05, **P < 0.01, ***P < 0.001 based on a Student’s t test, n = 4). c Confocal images of immunofluorescence staining of pHrodo-TDM1 and LAMP1 in NCIN87 cells in the presence and absence of lovastatin. Scale bars: 100 μm and 50 μm (inset). d Western blot of CAV1 and HER2 immunofluorescence in NCIN87 s.c. tumors from athymic nude mice. Lovastatin (8.3 mg/kg of mice) was orally administrated twice with an interval of 12 h between each administration. Scale bars: 50 μm. e Representative coronal PET images and TDM1-tumor uptake at 4, 8, 24, and 48 h p.i. of [⁸⁹Zr]Zr-DFO-TDM1 in athymic nude mice bearing s.c. NCIN87 tumors. Lovastatin (8.3 mg/kg of mice) was orally administrated 12 h prior and at the same time as the tail vein injection of [⁸⁹Zr]Zr-DFO-TDM1 (6.66–7.4 Mbq, 45–50 μg protein). Bars, n = 5 mice per group, mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 based on a Student’s t test. %ID/g, percentage of injected dose per gram. f [⁸⁹Zr]Zr-DFO-TDM1 uptake in HER2-expressing gastric PDXs containing varying levels of CAV1 and administered saline (blue color) or statin (red color). PDX IDs in this figure match patient IDs shown in Fig. 1. Points, n = 5 mice per group, mean ± S.E.M, **P < 0.01 based on a Student’s t test. %ID/g, percentage of injected dose per gram. g Cell viability of NCIN87 cells at 48 h after cells incubation with Trastuzumab (Trast) and TDM1 alone or in combination with lovastatin. Bars, n = 5–7 per group, mean ± S.E.M. *P < 0.05, **P < 0.01, based on a Student’s t test. h, i Western blots of HER2 signaling and quantification of NCIN87 cells after 48 h incubation with TDM1 alone or in combination with lovastatin. Bars, quantification of Western blots shown in Fig. 3i. Supplementary Fig. 13 shows quantifications of three independent assays. Source data are provided as a Source Data file.

Fig. 4. Lovastatin enhances TDM1 efficacy and Trastuzumab-mediated ADCC.

a–d Superior in vivo therapeutic efficacy of TDM1 combined with lovastatin when compared with TDM1 alone. a Intravenous TDM1 administration 5 mg/kg weekly (for 5 weeks) was started at day 0. Lovastatin (4.15 mg/kg of mice) was orally administrated 12 h prior to and simultaneously with the intravenous injection of TDM1. Lovastatin enhanced TDM1 efficacy of nu/nu female mice bearing NCIN87 gastric xenografts (b), and NSG mice bearing CAV1-high PDXs (d). *P < 0.05, **P < 0.01, ***P < 0.001 based on a Student’s t test (n = 8–10 mice per group). c Western blot analyses of AKT, ERK, Tyr, CAV1, and CREB protein expression and phosphorylation in NCIN87 xenografts at 40 days after treatment with lovastatin, TDM1, or TDM1/lovastatin. e NSG mice bearing NCIN87 xenografts were intravenously injected 1 × 10⁶ human NK cells at day 0. One day after NK cells tail vein injection, the IL-15/IL-15Rα complex was intraperitoneally administered at a dose of 1.25 μg/mouse. Trastuzumab or Trastuzumab/lovastatin efficacy was then evaluated during a cytokine-dependent NK expansion phase (week 1–week 3). Lovastatin enhanced Trastuzumab efficacy in NSG mice humanized with NK cells and bearing NCIN87 xenografts (n = 8–10 mice per group, mean ± S.E.M.). Statistical analyses performed using ANOVA coupled to Scheffé's method. f Trastuzumab/lovastatin efficacy is higher than the combination of Fc-silent Trastuzumab (Trastuzumab F(ab’)₂ fragments or deglycosylated Trastuzumab) in NSG mice humanized with NK cells and bearing NCIN87 xenografts (n = 8–10 mice per group, mean ± S.E.M.). Statistical analyses performed using ANOVA coupled to Scheffé's method. g Kaplan–Meier analysis of statin use and HER2-expressing GC disease outcome in patients treated with Trastuzumab. Patients without statin treatment (blue color, n = 27) have a worse survival than patients treated with statin (red color, n = 19). Log rank; p = 0.005. Source data are provided as a Source Data file.