Fig 2: NRF2 knockdown causes downregulation of HER2 and HER3 levels, repression of pAKT, and sensitisation to targeted immunotherapeutics. (a) Immunoblotting analysis showing inhibition of RTK signaling following depletion of NRF2 mRNA by SiRNA in SKOV3 cell line. Exponentially growing cells were either transfected with scrambled SiRNA (Scrmbl) or transfected with 75 pmol of NRF2 SiRNA for either 24 or 48 h or 100 pmol of NRF2 SiRNA for 48 h and processed for immunoblotting using relevant antibodies (Table 1). ß-actin was used as a loading control. Bar chart shows protein levels by quantifying immunoblot signal intensities obtained and normalised to the value of untreated (UT) control and expressed as fold change. (b) Immunofluorescent labelling of endogenous total HER2 or phospho-AKT exhibits repression following NRF2 knockdown. Cells were transfected as in (a) and processed for immunocytochemistry. Relevant primary antibodies were used to stain HER2 or phospho-AKT followed by Alexa Fluor conjugated secondary antibody (red fluorescence). Nuclear reference was provided by costaining with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Images were captured with Leica DMiRe2 electronic microscope at 100x objective while merging, colocalisation, and further analysis were performed by using integrated features of Andor iQ Core software (ANDOR Technologies Ltd.). Scale bar indicates 10 µm. (c and d) HER2 and HER3 downregulation following NRF2 knockdown is caused by their transcriptional repression. Exponentially growing PEO1 cells (c) or SKOV3 cells (d) were transfected with either empty PGL3 basic vector or 1 µg PGL3 basic vector with cloned 1.5 kb fragments of either HER2 (prHER2) or HER3 (prHER3) upstream promoter regions driving the expression of luciferase gene. Cotransfection with 0.2 µg pRL-CMV plasmid was performed as an internal transfection control. At 24 h after transfection, cells were either left untreated (UT) or treated with 200 µM tBHQ as indicated for 4 h following which, cells were processed for dual luciferase reporter assay (Promega) to record luciferase activity in multiplate reader (MODULUS, Promega). (d) The same was done for SKOV3 cell lines. (e) Knockdown of NRF2 through SiRNA sensitises cancer cell to RTK inhibitors while parallel knockdown of KEAP1 partially relieves this sensitisation. Cells were transfected with scrambled SiRNA or SiRNA targeting NRF2 either alone or with the inclusion of KEAP1 SiRNA. Following further 24 h incubation, cells were either left untreated or treated with 25 µg/mL of HER2 inhibitors Pertuzumab and Trastuzumab. Cytotoxicity assay was performed as in (a). In (c–e), data are the means with ±S.D. of triplicates and expressed as fold change with statistical significance determined by ONE WAY ANOVA followed by Tukey's post hoc test (for c and d), or Student's t-test (for e) according to the scale *: p < 0.05, **: p < 0.01, and ***: p < 0.001.

Fig 3: Treatment with Trastuzumab and/or Pertuzumab causes cytotoxicity and modulates expression of pHER2, pAKT, NRF2 and KEAP1 in ovarian cancer cell lines(A) Effect of antibodies on cell number. Cells were treated with Pertuzumab (PR), Trastuzumab (TR) or combination (COMB) of antibodies in the presence of 1nM Heregulin. Antibodies were used at 20µg/mL. After treatment, cell number was assessed by use of the MTT assay as described in Materials and Methods. Values are means with ± S.D of triplicates and expressed as fold decrease to HRG alone. Statistical significance was determined between untreated and treated groups by ONE WAY ANOVA followed by Tukey's post hoc test. Asterisks indicate significance of difference between HRG and individual treatment group whereas “#” indicates significance of difference between combination and Pertuzumab (red sign) or combination and Trastuzumab (blue sign) according to the scale * P <0.05, **P <0.01, ***P <0.001. (B) Western blot analysis showing levels of phospho HER2 T877 and phospho AKT S487 and (C) NRF2 and KEAP1 in response to treatments with 20µg/mL of Pertuzumab (Pr), Trastuzumab (Tr) and their combination (Comb) in PEO4, OVCAR4 and SKOV3 cells. Exponentially growing cells were treated with media containing 1nM Heregulin alone, or with co-treatment of Pertuzumab, Trastuzumab or their combination for 96h before being analyzed as described in Materials and Methods. The bar charts show phospho HER2 or NRF2 (red bars) and phospho AKT levels or KEAP1 (blue bars) following quantification of the immunoblot signal intensities obtained and normalized to HRG treatment group and expressed as fold change. The signal intensities of bands were quantified through integrated optical densitometry measurement using Gelpro software (Version 3.1, Media Cybernetics).

Fig 4: Treatment with HER2 targeting immunotherapeutic agents causes induction of HDAC1 expression and repression in phospho-HDAC levels(A) Immunoblotting analysis showing induction of total HDAC 1 levels following treatment with combination of Pertuzumab and Trastuzumab in PEO4, OVCAR4 and SKOV3 cell lines. Exponentially growing cells were either left untreated (UT) or treated with media containing 1nM Heregulin alone (HRG), or with co-treatment of 20µg/mL of Pertuzumab (PR), Trastuzumab (TR) or their combination (COMB) for 96 h before being harvested for immunoblotting using an anti-HDAC1 antibody (Table 1). Ponceau stain of the same blot was used as loading control Red bars indicate HDAC1 levels following quantification of the immunoblot signal intensities obtained and normalized to the value of UT and expressed as fold change. The signal intensities of bands were quantified as described in Materials and Methods, (B) Treatment with HER2 inhibitors causes induction of nuclear HDAC1 levels. Immunofluorescent labelling of endogenous HDAC1 in cells previously grown on poly-L lysine coated coverslips and exposed to treatments as in (A). For immunolabelling, Alexa Fluor® 568 conjugated secondary antibody (red fluorescence) was used. Nuclear reference was provided by co-staining with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Images were captured with Leica DMiRe2 electronic microscope using integrated features of ANDOR iQ core software (ANDOR Technologies Ltd). Scale bar indicates 10µm. These are representative images taken in different field of views with relevant fluorescence channels and 100x objective. (C) Immunoblotting analysis of phospho-HDAC levels following treatment with combination of Pertuzumab and Trastuzumab in PEO4, OVCAR4 and SKOV3 cell lines. Exponentially growing cells were either left untreated (UT) or treated with media containing 1nM Heregulin alone (HRG), or with co-treatment of 20µg/mL of Pertuzumab (PR), Trastuzumab (TR) or their combination (COMB) for 96 h before being harvested to prepare protein lysates and processed for immunoblotting using an anti-phospho HDAC antibody (Table 1). Ponceau stain of the same blot was used as loading control (not shown). Red bars indicate phospho-HDAC levels following quantification of the immunoblot signal intensities obtained in (A) and normalized to the value of UT and expressed as fold change. The signal intensities of bands were quantified through integrated optical densitometry measurement as described in Materials and Methods.

Fig 5: The binding domains of KRT19 and HER2 identified by the co-transfection of KRT19 and HER2.(A) The truncated variants of KRT19 were constructed. The detail is as follows: Full, full length of KRT19; T1, head, coil 1 A, coil 1B and coil 2; T2, head, coil 1 A and coil 1B; T3, head and coil 1 A; T4, head; T5, coil 1 A, coil 1B, coil 2 and rod-like helical tail; T6, coil 1 A and coil 1B; and T7, coil 2 and rod-like helical tail. (B) HER2 and each variant of KRT19 were transfected to HEK293T. HER2 complexes were collected using anti-HA tag immunoprecipitation. Only T4-including variants of KRT19 were able to bind to HER2. (C) The truncated variants of HER2 were constructed. The detail is as follows: Full, full length of HER2; Ex, extracellular domain; KD, kinase domain; and C-ter, COOH-terminal domain. (D) KRT19-T4 and each truncated variant of HER2 were co-transfected into HEK293T. These transfected cell lysates and their culture media were immunoprecipitated with anti-HA-tag beads. KRT19 only binds to intracellular HER2. Full length and fragments of HER2 were tagged with HA, and full length and fragments of KRT19 were tagged with Flag. Full-length blots are presented in Supplementary Fig. S9.