Mixed lineage kinase 3 and CD70 cooperation sensitize trastuzumab-resistant HER2+ breast cancer by ceramide-loaded nanoparticles

Abstract

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2⁺) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2⁺ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2⁺ human breast tumors. Our in vitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2⁺ breast cancer.

Keywords: CD70; MLK3; apoptosis; breast cancer; trastuzumab resistance.

Fig. 1.

MLK3 expression and activity are diminished in TR breast cancer cells and tumors. (A and B) Western blot of MLK3 in whole-cell lysates, isolated fromTS and TR SKBR3 and BT-474 cell lines. GAPDH was taken as a loading control. (C–F) In vitro MLK3 kinase activity and Western blotting of phospho (p-SEK1) and total SEK1, and phospho (p-c-Jun) and total c-Jun in whole-cell lysates, isolated from control and trastuzumab-treated, TS and TR SKBR3 and BT-474 cell lines. (G) Representative images of phospho MLK3 (pMLK3) IHC staining and quantification in tumor sections from trastuzumab responders and nonresponders human breast cancer tissues (n = 10/group). (Scale bar: 20 μm.) Data represent the mean ± SD; **P < 0.001, Student’s t test.

Fig. 2.

Ceramide-loaded nanoparticles (PPP-CNP) regulate MLK3 and its downstream signaling. (A) Direct binding of ceramide with recombinant baculoviral-MLK3 protein using large multilamellar vesicle (LMV) assay. (B) The hydrophobic interactions between POEG-b-PPPMP polymer and ceramide. (C) Flow cytometry analyses for cellular uptake of rhodamine B loaded G-PPP or PPP-CNP nanoparticles (RB-PPMP) in BT-474-TR cells (n = 3). (D and E) Flow cytometry analyses of ceramide levels in control, G-PPP-, and PPP-CNP-treated BT-474-TR and SKBR3-TR cells (n = 3). (F and G) Flow cytometry analyses of MLK3 activity (i.e., phospho-MLK3) in control, the ghost (G-PPP), and ceramide nanoparticles (PPP-CNP)-treated BT-474-TR and SKBR3-TR cell lines (n = 3). (H and I) Western blotting of phospho-JNK (p-JNK) and total JNK (JNK); p-c-Jun and c-Jun in TR cell lines, treated either with G-PPP or PPP-CNP. GAPDH was taken as a loading control. (J) AKT phosphorylation array and densitometry in control, G-PPP-, and PPP-CNP-treated SKBR3-TR cells. Data represent the mean ± SD; ***P < 0.0001. One-way ANOVA.

Fig. 3.

PPP-CNP inhibits cellular proliferation and induces apoptosis via MLK3 and AKT pathways in TR cells. (A) BrdU and 4′,6-diamidino-2-phenylindole (DAPI) staining for cell-cycle analyses in BT-474-TR cells treated with G-PPP or PPP-CNP (n = 3). (B) Cell death and apoptosis analyses in BT-474-TR cells treated with G-PPP or PPP-CNP (n = 3). (C–E) Western blotting for Bax, Bcl-2, cleaved PARP (c-PARP), FAS, and FAS-L in whole-cell lysates from G-PPP- or PPP-CNP-treated BT-474-TR cells. GAPDH was taken as a loading control. (F) Flow cytometry analysis of Annexin-V expression in control and treated parental and MLK3-KD BT-474-TR cells (n = 3). (G) Flow cytometry analysis of Annexin-V expression in control and treated parental and Myr-AKT expressing BT-474-TR cell line (n = 3). (H) Western blotting with M2 flag-tag and MLK3 antibody for doxycycline-inducible MLK3 (S674A) and MLK3 expression in a stable BT-474-TR cell line. (I) Flow cytometry analysis of Annexin-V expression in control and Dox-treated BT-474-TR cell line, expressing MLK3 (S674A) (n = 3). (J) Percent viability of BT-474-TR cells upon treatment with G-PPP or PPP-CNP in the presence and absence of trastuzumab (n = 2). Data represent the mean ± SD *P < 0.05, **P < 0. 001 and ***< 0.0001. One-way ANOVA (A and B) and Student’s t test (F, G, I, and J).

Fig. 4.

PPP-CNP regulates CD70 gene and protein expression via MLK3 in TR cells. (A) RT² profiler PCR array for apoptosis-related gene expressions in control, G-PPP-, and PPP-CNP-treated BT-474-TR cells (n = 2). (B) Western blot for CD70 and CD27 in BT-474-TR and SKBR3-TR cells treated with G-PPP or PPP-CNP. GAPDH was taken as a loading control. (C) Released CD70 in BT-474-TR cell culture media treated with vehicle control or G-PPP or PPP-CNP, estimated by human CD70-specific ELISA (n = 3). (D and E) Flow cytometry analyses of CD70 surface expression on control and treated parental and MLK3-KD BT-474-TR and SKBR3-TR cells (n = 3). (F) Flow cytometry analyses of CD70 expression in BT-474-TR cells upon treatment with G-PPP or PPP-CNP in the presence and absence of AP1 and NF-κB dual inhibitor, SP100030 (n = 3). (G) Western blotting of total and phospho-NF-κB in BT-474-TR cells treated with G-PPP or PPP-CNP. (H) Immunofluorescence staining of pMLK3 and pNF-κBp65 in BT-474-TR cells treated with G-PPP or PPP-CNP. (Scale bar: 50 µm.) Data represent the mean ± SD; ***P < 0.0001. One-way ANOVA (C and F) and Student’s t test (D and E).

Fig. 5.

PPP-CNP induces apoptosis in TR breast cancer cells via the MLK3–CD70 axis. (A) Flow cytometry analyses of apoptosis (Annexin V⁺) and (B) cleaved caspase-3 (c-Casp3) in CD70⁺ and CD70⁻ BT-474-TR cell populations (n = 3). (C) CD70 protein expression in BT-474-TR cells transfected with either scrambled (siControl) or CD70-specific siRNA (siCD70). GAPDH was taken as a loading control. (D) Flow cytometry analyses for apoptosis (Annexin V) in siControl and CD70-knockdown BT-474-TR cells (n = 3). (E) Apoptosis (Annexin V expression) analyses by flow cytometry in BT-474-TR cells pretreated with either isotype immunoglobulin G or anti-CD27 antibody followed by treatment with G-PPP or PPP-CNP (n = 3). (F) Flow cytometry analyses of CD70⁺Annexin V⁺ cell populations and (G–I) CD70⁺c-Cas9⁺, CD70⁺c-Cas8⁺, and CD70⁺c-Cas3⁺ in parental and MLK3-KD BT-474-TR cell populations upon treatment with either G-PPP or PPP-CNP (n = 3). Data represents the mean ± SD; **P < 0.001 and ***P < 0.0001. Student’s t test (A, B, D, and F–I) and one-way ANOVA (E).

Fig. 6.

CD70 expressions and NF-κBp65 activity are diminished in TR cells and patient tumors. (A–C) Flow cytometry analyses for CD70 expression in TS and TR SKBR3 and BT-474 and 4T1.HER2 and 4T1.HER2-TR cell lines (n = 3). (D) Representative images of CD70 immunofluorescence staining in tumor sections from trastuzumab responder and nonresponder human breast cancer tissues (n = 4/group). (Scale bar: 5 µm.) (E) Representative images of p-NF-κBp65 IHC staining and scoring in trastuzumab responder and nonresponder human breast tumor tissues (n = 9/group). (Scale bar: 10 µm.) Data represent the mean ± SD; *P < 0.05, and **P < 0.001, Student’s t test.

Fig. 7.

PPP-CNP induces apoptosis in TR PDOs and reduces TR PDX tumor burden. (A) Representative immunofluorescent images (Left) and quantification (Right) of PDOs-TS and PDOs-TR treated either with G-PPP or PPP-CNP and labeled with TUNEL (n = 5–8 PDOs/group). DAPI was used for nuclear staining. (Scale bar: 2000 µm.) (B) Viability of normal human liver organoids treated either with G-PPP or PPP-CNP. Acetaminophen was used as a positive control (n = 3). (C) Tumor volume of saline control, G-PPP-, and PPP-CNP-treated groups (n = 4 mice/group). (D) Ki67 IHC staining and nuclear Ki67⁺ count in tumor sections from control and treated mice. (Scale bar: 10 µm.) (E) TUNEL assay and TUNEL⁺ cell count in control, G-PPP-, and PPP-CNP-treated tumor sections. Propidium iodide was used for nuclear staining, (Scale bar: 50 µm.) Data represent the mean ± SD; *P < 0.05, **P < 0.001, and ***P < 0.0001, one-way ANOVA. ##<0.001 and ###<0.0001 (G-PPP vs. PPP-CNP), Student’s t test.