Strategic Trastuzumab Mediated Crosslinking Driving Concomitant HER2 and HER3 Endocytosis and Degradation in Breast Cancer

Efficacious anticancer therapies for targeting plasma membrane receptors with antibody based therapeutics are often contingent on sufficient endocytic delivery of receptor and conjugate to lysosomes. This results in downregulation of receptor activity and, in the case of antibody-drug conjugates (ADCs), intracellular release of a drug payload. The oncogenic receptor HER2 is a priority therapeutic target in breast cancer. Known as an “endocytosis resistant” receptor, HER2 thwarts the receptor downregulating efficiency of the frontline treatment trastuzumab and reduces the potential of trastuzumab-based therapies such as trastuzumab-emtansine. We previously demonstrated that strategically inducing trastuzumab and HER2 crosslinking in breast cancer cells promoted endocytosis and lysosomal delivery of the HER2-trastuzumab complex, stimulating downregulation of the receptor. Here we reveal that HER3, but not EGFR, is also concomitantly downregulated with HER2 after crosslinking. This is accompanied by strong activation of MEK/ERK pathway that we show does not directly contribute to HER2/trastuzumab endocytosis. We show that crosslinking induced trastuzumab endocytosis occurs via clathrin-dependent and independent pathways and is an actin-dependent process. Detailed ultrastructural studies of the plasma membrane highlight crosslinking-specific remodelling of microvilli and induction of extensive ruffling. Investigations in a cell model of acquired trastuzumab resistance demonstrate, for the first time, that they are refractory to crosslinking induced HER2 endocytosis and downregulation. This implicates further arrest of HER2 internalisation in developing trastuzumab resistance. Overall our findings highlight the potential of receptor crosslinking as a therapeutic strategy for cancer while exposing the ability of cancer cells to develop resistance via endocytic mechanisms.

centrifuged at 13,000 x g for 10 min at 4 °C and the supernatant collected. Immunoprecipitation was performed using published protocol (58) adapted and outlined below.
Generation of IP beads: anti-HER2 mAb 7C2 (BSA/glycerol free) was purchased from Insight Biotechnology (Wembley, UK) and diluted to 400 µg/mL in 0.4 M Sodium Citrate, 0.1M Sodium HEPES, pH7.5. All 100 µg 7C2 was added to 5 mg Ultralink Biosupport (Fisher Scientific) and allowed to react for a minimum of 3 hr at room temperature under constant rotation. 7C2 beads were pelleted at 1,200 x g for 5 min and the supernatant removed. To both 7C2 beads and 5 mg control beads 10X the bead swell volume (10xSV) of 2M glycine in PBS was added and the solution allowed to react for 3 hr at room temperature under rotation. 7C2 and control beads were washed by pelleting (1,200 x g, 5 min) the beads and resuspended in 10xSV PBS for 15 min at room temperature under rotation. This was followed by further washes in 10xSV 1M NaCl, and two 10xSV PBS washes.
The beads were finally resuspended in 200 µL PBS and kept at 4°C for a maximum of 24 hr.
Immunoprecipitation using Ultralink Biosupport beads: 100 µg of cell lysate was incubated with 15 µL of 7C2 or control beads in a final volume of 200 µL lysis buffer overnight at 4 °C under constant rotation. The beads were pelleted at 1,200 x g for 5 min and the pellet was washed twice with 500 µL lysis buffer. The final pellet was resuspended in 15 µL PBS prior to adding SDS loading buffer containing DTT (5 µL). The sample was boiled for 4 min at 96 °C before being separated on a 7.5% SDS-PAGE gel, transferred to PDVF membranes and probed for EGFR, HER3 and finally HER2 as per the method section. were either untreated (control) or incubated with Tz diluent control 30 min followed by SA alone for 1 hr, Tz alone for 30 min (+ 1 hr SA diluent control), or Tz for 30 min followed by SA for 1 hr (Tz+SA). For the 3 hr time point cells were subject to a 2 hr chase in CIM before lysate collection. Cell lysates were collected from three independent experiments, representative blots are shown. Western blotting was performed for total HER2, P-ERK (Thr202/Tyr204) and total ERK. B) Band intensities were quantified using ImageJ software. Mean from 3 independent experiments is shown, error bars represent SE, *p≤0.05. Data show ERK activation and slight reduction in HER2 following crosslinking challenge over 3 hr time course, ERK was also activated by HER2 but there was evidence of this beginning to subside. C) SKBR3 cells and BT474 cells were either untreated (control) or incubated with Tz diluent control 30 min followed by SA alone for 1 hr, Tz alone for 30 min (+ 1 hr SA diluent control), or Tz for 30 min followed by SA for 1 hr (Tz+SA) then a 6 hr chase in CIM. Cell lysates were collected from three independent experiments, representative blots are shown. Western blotting was performed for P-HER2 Tyr1248, P-MEK (Ser217/221) and total MEK. D) Band intensities were quantified using ImageJ software and mean from 3 independent experiments is shown (Error bars represent SEM). The data demonstrate that crosslinking induced MEK activation of varying magnitude. cells is not significantly altered 72 hr after Avi-Bi-RAC treatment. Cells were either untreated (control) or incubated with Tz diluent control 30 min followed by SA alone for 1 hr, Tz alone for 30 min (+ 1 hr SA diluent control), or Tz for 30 min followed by SA for 1 hr (Tz+SA). Cells were incubated in IM for 72 hr post-crosslinking.

SUPPLEMENTARY FIGURE
Cell Viabilities were analysed by CellTiter-Blue TM assay. Viability was not significantly altered by any of the treatments compared with untreated control cells. B) HER2 levels in Avi-bi-RAC treated SKBR3 breast cancer cells recover in 48 hr, after initial downregulation at 6 and 24 hr post-treatment. SKBR3 cells were either untreated (control) or incubated with Tz alone or Tz followed by SA. The cells were subjected to a posttreatment time course of 6-48 hr in IM. Cell lysates were collected Western blotting was performed for HER2 and β-tubulin. C) Band intensities were quantified using ImageJ software and converted to graphs in Excel. The