Fig 2: Lapatinib increased HER2 levels by elevating protein synthesis while dacomitinib prevented this effect. a qPCR showing the effects of lapatinib on EGFR, HER2 or ErbB3 mRNA. 22Rv1 cells were cultured in serum-containing media with 2 µM lapatinib or dacomitinib before being lysed with TRIZOL reagent as per the manufacturer’s instructions. Results represent mean ± S.D. of experiments performed in triplicate biological replicates. b 22Rv1 cells were treated for 2 or 8 h with vehicle or 2 µM lapatinib with or without 5 µg/ml MG-132 and fractionated as described. c Cells were pre-treated for 3 h with 100 µg/ml of cycloheximide before being washed off and allowed to recover for 24 h in fresh media containing either vehicle (DMSO) or 2 µM lapatinib. Fractionation was performed as detailed. d Dacomitinib decreases protein stabilities of EGFR, HER2 and ErbB3 despite causing an increase in EGFR and ErbB3 mRNA. Cells were plated and allowed to attach overnight at 37 °C before being pre-treated for 3 h with 100 µg/ml of cycloheximide dissolved in 100% DMSO or an equivalent volume of 100% DMSO. Cells were subsequently washed twice with 1X phosphate-buffered saline (PBS) and allowed to recover in fresh media containing either 2uM dacomitinib or an equivalent volume of 100% DMSO. Cells were collected, and lysates prepared at 2, 8 or 24 h for analysis by western blotting

Fig 3: MUC4 up-regulates EGFR family receptors and induces downstream Erk1/2 and PKC-? pathways.(A) Immunoblot analyses showed reduced expression of EGFR, ErbB2, and ErbB3 in MUC4 knockdown cells compared with control cells. (B) Reduced expression (using immunoblot) of Sprouty 2 was detected in MUC4 knockdown cell when compared with control cells. (C) Immunoblot showed that reduced phosphorylation of Erk1/2 and expression of PKC-? in MUC4 knockdown cells compared with control cells. ß-actin was used as a loading control. (D) Immunoblot analyses showed reduced expression of ß-catenin and its target gene product cyclin D1in MUC4 knockdown compared with control cells.

Fig 4: Anti-tumor effect of HER3 inhibition in syngeneic HNSCC models and increased durable responses to PD-1 blockade.a C57Bl/6 mice were implanted with 1 × 106 of 4MOSC1 cells into the tongue. After tumors reached ~30 mm3, mice were treated IP with of isotype control, CDX-3379 (20 mg/kg), anti-PD-1 (10 mg/kg), or a combination of CDX-3379 and PD-1 three times per week for 3 weeks. Individual growth curves of 4MOSC1 tumor-bearing mice are shown (n = 10 per group). b C57Bl/6 mice were implanted with 2 × 106 MOC1 cells into the flanks. After tumors reached approximate 50 mm3, mice were treated same as panel (a). Individual growth curves of MOC1 tumor-bearing mice are shown (n = 8 per group). c A Kaplan–Meier curve showing the survival of mice from panels (a) and (b). The death of animals occurred either naturally, when tumor compromised the animal welfare, when tongue tumor volume (panel a) reached 100 mm3 (n = 10 mice per group), or when flank tumor volume (panel b) reached 500 mm3 (n = 8 mice per group). Two sided log-rank/Mantel–Cox test. d Representative immunohistochemical analysis of pS6 and BrdU in the short-term treatment groups (every other day for three treatments) from panel (a). Brown chromogen deposition reflects the immunoreactivity; hematoxylin was used as a nuclear counterstain (blue). Scale bars represent 25 µm. Quantification from images on the left using Qupath software and the percentage of positive staining are shown on each image. e Immunofluorescent staining of CD8 and CK5 in the short-term treatment from panel (a). Source data are provided as a Source Data file.

Fig 5: Dacomitinib, unlike lapatinib, disrupts membrane localisation, decreases EGFR-HER2 dimerisation and downstream signalling of EGFR family members in CRPC cells. a In 22Rv1 cells, pulldown assays demonstrate the significant disappearance of EGFR-HER2 dimers with dacomitinib and, to a lesser extent, HER2-ErbB3 dimers. Cells were cultured in the presence of DMSO, 2 µM lapatinib or 2 µM dacomitinib for 72 h, at the end of that period, they were further treated with PBS, 10 ng/ml EGF or 50 ng/ml HRG for 30 min. Note that in these cells, an upper non-specific band (narrow arrow) appeared in the immunoprecipitated lysates in addition to the RTK band (thick arrow) that was not apparent in regular Westerns. b Immunofluorescent microscopy indicating the loss of EGFR and HER2 staining in dacomitinib but not lapatinib-treated cells. 22Rv1 cells were treated with DMSO, 2 µM lapatinib or 2 µM dacomitinib for 72 h, then fixed and stained with anti-EGFR antibody (secondary—Rhodamine-tagged) and anti-HER2 (secondary—FITC tagged) as well as DAPI (blue). Merged pictures depict colocalisation. c 22Rv1 (upper) or C4-2 (bottom) cells were cultured in androgen-containing media and treated with 2uM lapatinib or dacomitinib for 72 h. Hundred percent DMSO was used as the control. (upper) Lapatinib treatment results in prominent EGFR and ErbB3 membrane staining. Dacomitinib treatment results in the disappearance of EGFR and ErbB3 protein. Cell numbers also appeared greatly reduced. (bottom) Dacomitinib also decreases the staining intensity of HER2 and EGFR in C4-2 cells. d Western blots comparing the effects of dacomitinib and lapatinib on phosphorylated p44/42 ERK1/2 (Thr202/Tyr204) and phosphorylated AKT (Ser473). e Immunofluorescent imaging showing that dacomitinib alters EGFR membrane staining but does not disrupt the membrane itself. Cells were stained with anti-E-cadherin (rhodamine tagged) and EGFR (FITC tagged). In control cells, EGFR colocalised with E-cadherin (yellow merge) showing that both proteins were expressed in the plasma membrane. Upon treatment with 2 µM lapatinib, the colocalisation intensified, showing increased localisation of EGFR in the membrane. On the other hand, 2 µM dacomitinib decreased EGFR levels in the membrane but left E-cadherin levels intact, demonstrating that membrane integrity was not compromised. Pictures were magnified at ×100