Overcoming brain-derived therapeutic resistance in HER2+ breast cancer brain metastasis

Abstract

Brain metastasis of HER2+ breast cancer occurs in about 50% of all women with metastatic HER2+ breast cancer and confers poor prognosis for patients. Despite effective HER2-targeted treatments of peripheral HER2+ breast cancer with Trastuzumab +/-HER2 inhibitors, limited brain permeability renders these treatments inefficient for HER2+ breast cancer brain metastasis (BCBM). The scarcity of suitable patient-derived in-vivo models for HER2+ BCBM has compromised the study of molecular mechanisms that promote growth and therapeutic resistance in brain metastasis. We have generated and characterized new HER2+ BCBM cells (BCBM94) isolated from a patient HER2+ brain metastasis. Repeated hematogenic xenografting of BCBM94 consistently generated BCBM in mice. The clinically used receptor tyrosine kinase inhibitor (RTKi) Lapatinib blocked phosphorylation of all ErbB1-4 receptors and induced the intrinsic apoptosis pathway in BCBM94. Neuregulin-1 (NRG1), a ligand for ErbB3 and ErbB4 that is abundantly expressed in the brain, was able to rescue Lapatinib-induced apoptosis and clonogenic ability in BCBM94 and in HER2+ BT474. ErbB3 was essential to mediate the NRG1-induced survival pathway that involved PI3K-AKT signalling and the phosphorylation of BAD at serine 136 to prevent apoptosis. High throughput RTKi screening identified the brain penetrable Poziotinib as highly potent compound to reduce cell viability in HER2+ BCBM in the presence of NRG1. Successful in-vivo ablation of BCBM94- and BT474-derived HER2+ brain tumors was achieved upon two weeks of treatment with Poziotinib. MRI revealed BCBM remission upon poziotinib, but not with Lapatinib treatment. In conclusion, we have established a new patient-derived HER2+ BCBM in-vivo model and identified Poziotinib as highly efficacious RTKi with excellent brain penetrability that abrogated HER2+ BCBM brain tumors in our mouse models.

Keywords: ErbB inhibitors; HER2; Lapatinib; Poziotinib; brain metastasis; brain tumor remission; breast cancer; neuregulin-1.

Figure 1.. HER2+ BCBM94 cells establish hematogenic brain metastasis in mice.

(A) BCBM94 cells produce hematogenous brain metastasis upon intracardiac xenografting in RAG2yc−/− mice. H&E and IHC staining for Ki67+ nuclei and CD31+ endothelial cells in FFPE mouse brain tissues containing BCBM94 lesions. Magnification 200x (B,C) BCBM94 cells retain HER2 expression in-vivo and in-vitro. HER2 IHC staining of FFPE tissue sections of patient’s breast, patient’s brain, and mouse brain containing BCBM94 lesions. Magnification 200x. ICC staining of cultured formaldehyde-fixed BCBM94 cells show strong membrane expression of HER2 (ErbB2). (D) BCBM94 is a Luminal-B HER2+ BC model. Western blot (WB) comparing total protein expression of HER2 (ErbB2), ERα, and PRα/β in the triple-negative MDA-MB-BR and HER2+ BCBM94, BT474, and SKBR3 cell lines in-vitro. (E) NRG1 is expressed in the TME of BCBM94 brain metastases. In-situ expression of NRG1 and ErbB2 mRNA in the FFPE mouse brain tissue containing BCBM94 metastases was assessed with the RNAscope 2.5 HD Duplex assay. Black arrows indicate NRG1 mRNA (red dots) within non-tumoral cells in the mouse brain. NRG1+ cells are abundantly present in various regions of the mouse brain, including the TME of HER2+ BCBM94 (blue) metastasis. Magnification 200x. (F) HER2+ BC cell models are devoid of NRG1. WB compared NRG1 protein expression in the triple-negative MDA-MB-BR and HER2+ BCBM94, BT474, and SKBR3 cell lines in-vitro.

Figure 2.. NRG1 rescues BCBM94 cells from Lapatinib-induced cytotoxicity.

(A-D) Cell viability of BCBM94 under Lapatinib (Lap) +/− rhNRG1 treatment was assessed in the WST-1 assay. The endpoint absorbance readouts were used for quantification of the relative cell viability (mean +/− SD, n=3) (A-C). Cell proliferation and colony formation potential of BCBM94 cells under Lap +/−rhNRG1 treatment were assessed in colony formation assays (D). The bar chart presents the average number of colonies formed by BCBM94 cells under Lap +/− rhNRG1 treatment (n=2) (D). Representative images of colonies are shown below the plot (D). (E-I) NRG1 counteracts Lapatinib-induced apoptosis. The chart presents cleaved / full length PARP protein ratio in BCBM94 cells under Lap +/−rhNRG1 conditions measured by Western blot (WB) and quantified with densitometry (n=3) (E). A representative WB is shown below the plot (E). WB detection of cleaved and full-length PARP proteins in BT474 cells is shown under Lap +/− rhNRG1 conditions (n=1) (F). Relative luminescence values represent the activity of caspase-3 and caspase-7 under Lap +/−rhNRG1 conditions measured using a CaspaseGlo 3/7 assay (mean +/− SD, n=3) (G). Detection of the cleaved/ pro- caspase-9 protein ratio in BCBM94 cells under Lap +/−rhNRG1 conditions was measured by WB and quantified with densitometry (n=3) (H). A representative WB is shown below the chart (H). WB detection of cleaved / pro- caspase-9 proteins in BT474 cells under Lap +/− rhNRG1 conditions (n=1) (I). Bar charts present mean +/− SD,*p<0.05, **p<0.01.

Figure 3.. Anti-apoptotic actions of NRG1 involve BCL2 proteins

(A,B) NRG1 rescues Bad phosphorylation under Lapatinib (Lap). The phospho−/ total Bad protein ratio in BCBM94 cells under Lap +/−rhNRG1 conditions was determined by WB and quantified with densitometry (n=3) (A). A representative WB is shown below the chart (A). WB images show expression of phospho- and total Bad proteins in BT474 cells under Lap +/− rhNRG1 conditions (n=1) (B). (C) NRG1 protects mitochondria from Lapatinib-induced damage. Relative fluorescence intensity values represent the number of active mitochondria in BCBM94 cells under Lap +/−rhNRG1 conditions detected with MitoTracker^® (n=2). Representative IF images are shown below the graph, magnification 200x (C). (D) NRG1 prevents aggregation of the mitochondria outer membrane pore-formers Bax and Bak under Lapatinib. BCBM94 cells treated with Lap +/−rhNRG1 were PFA-fixed for detection of Bak and Bax by ICC/IF. The white arrow indicates punctate Bax aggregates co-localizing with Bak under Lapatinib treatment (n=2). Magnification 630x. Bar charts present mean +/− SD, *p<0.05, **p<0.01.

Figure 4.. NRG1 rescues ErbB3 phosphorylation under HER2 inhibition in BCBM cells

(A-D) NRG1 rescues ErbB3 phosphorylation under Lapatinib (Lap). Protein ratios for phospho−/ total ErbB2 and phospho−/ total ErbB3 were determined in BCBM94 cells under Lap +/−rhNRG1 by WB and quantified with densitometry (n=3) (A). Total ErbB1 (EGFR) and total ErbB3 protein levels in BCBM94 cells under Lap +/−rhNRG1 conditions were measured by WB and quantified with densitometry (n=3) (B). Representative WBs are shown below the charts (A, B). WB images show the detection of phosphor-ErbB1–4 and total ErbB1–4 proteins in BT474 cells under Lap +/−rhNRG1 conditions (n=1) (C,D). (E) Phosphorylated ErbB3 is expressed by BCBM94 tumors in-vivo. IHC analysis of patient’s breast, patient’s brain, and mouse brain FFPE tissue sections containing BCBM94 lesions, magnification 200x. Bar charts present mean +/− SD, *p<0.05, **p<0.01.

Figure 5.. ErbB3 signaling mediates anti-apoptotic actions of NRG1 under Lapatinib

(A, B) Knockdown of ErbB3 increases PARP cleavage under Lapatinib (Lap) and mitigates NRG1 rescue. The cleaved/ full-length PARP protein ratio in BCBM94 cells under Lap +/−rhNRG1 +/−ErbB3siRNA conditions was measured by WB and quantified with densitometry (n=3) (A). A representative WB is shown below the chart (A). Protein levels of cleaved and full-length PARP proteins in BT474 cells under Lap +/−rhNRG1 +/−ErbB3siRNA conditions are shown by WB (n=1) (B). (C, D) Knockdown of ErbB3 attenuates rhNRG1-mediated rescue of phospho-Bad under combined Lap/ rhNRG1 treatment. The graph presents phospho−/ total Bad protein ratio in BCBM94 cells under Lap +/−rhNRG1 +/−ErbB3siRNA conditions measured by WB and quantified with densitometry (n=3) (C). A representative WB is shown below the chart (C). Protein levels of phospho- and total Bad proteins in BT474 cells under Lap +/−rhNRG1 +/−ErbB3siRNA conditions were detected by WB (n=1) (D). (E, F) NRG1 rescues expression and phosphorylation of Akt under Lapatinib. WB images show expression of phospho- and total Akt proteins in BCBM94 (representative examples, n=3) (E) and BT474 (n=1) (F) cells under Lap +/−rhNRG1 +/−ErbB3siRNA conditions. (G, H) The anti-apoptotic action of NRG1 is mediated through Akt. The graph presents phospho−/ total Bad protein ratio in BCBM94 cells under Lap +/−rhNRG1 treatment measured by WB and quantified with densitometry; the PI3K inhibitor PI-103 was used at 10 μM (G). Representative WB images are shown below the chart (n=3) (G) and present the levels of phospho−/ total-Akt and cleaved/ full-length PARP proteins in BCBM94. Protein levels of phospho−/ total-Akt, cleaved/ full-length PARP and phospho−/ total Bad proteins in BT474 cells under Lap +/−rhNRG1 exposure and treatment with PI-103 were determined by WB (n=1). Bar charts present mean +/− SD, **p<0.01

Figure 6.

Schematic illustration of the NRG1 actions that rescue Lapatinib-induced apoptosis in HER2+ BCBM cells.

Figure 7.. Poziotinib-induced cytotoxicity is not diminished by NRG1

(A) Fifty ErbB inhibitors were tested for differential sensitivity in BCBM94 versus HME1. 53 samples (3 compounds were represented by two separate batches) were tested at 22 concentrations ranging from 0.003pM to 40uM (BCBM94 n=4, HME1 n=1). The area under the curve (AUC, y-axis) of the non-linear fitting of the dose-response data was calculated as a metric to compare differential sensitivity to the compounds. The potency of each compound in the BCBM94 model is indicated on the x-axis. Compounds in the lower left corner of the plot are the most potent and BCBM94-selective. (B) Poziotinib is highly efficacious in reducing cell viability of both BCBM94 and BT474 HER2+ BC models. ErbB inhibitors were tested at concentrations ranging from 0.003 pM to 40uM and viability was measured using a CellTiterGlo assay after 72 h (BCBM94 n=4, BT474 n=1). (C) rhNRG1 reduces the cytotoxic activity of many ErbB inhibitors. The 50 ErbB inhibitors were tested +/− 5ng/mL rhNRG1 and viability was measured after 72h using CellTiterGlo. The differential sensitivity was assessed by comparing IC50 values (ratio) under both conditions (BCBM94 n=3, BT474 n=1). Data points that fall near the intersection of the dotted lines, including Poziotinib (Poz), represent compounds with equipotent activity +/− NRG1. Compounds to the upper right of the plot are those where NRG1 reduced the cytotoxic effect of the compound. (D-F) rhNRG1 failed to rescue tested HER2+ BC models from Poziotinib-mediated cytotoxicity. Cell viability of BCBM94 (n=3) (D, E) and BT474 (n=3) (F) under Poz +/−rhNRG1 treatment was assessed in the WST-1 assay. The endpoint absorbance readouts were used for quantification of the relative cell viability. (G, H) rhNRG1 failed to counteract Poziotinib-induced apoptosis. WB images show the levels of cleaved/ full-length PARP in BCBM94 (representative examples, n=3) (G) and BT474 (n=1) (H) cells under Poz +/−rhNRG1 treatment. (I, J) rhNRG1 was unable to rescue ErbB3 phosphorylation under Poziotinib. Representative WB images show the levels of phospho−/ total ErbB3 in BCBM94 (n=3) (I) and BT474 (n=1) (J) cells under Poz +/−rhNRG1 treatment. (K, L) rhNRG1 failed to rescue Akt phosphorylation under Poziotinib. Representative WB images show the levels of phospho−/ total ErbB3 in BCBM94 (n=3) (K) and BT474 (n=) (L) cells under Poz +/−rhNRG1 treatment. Bar charts present mean +/− SD, *p<0.05; **p<0.01.

Figure 8.

Schematic illustration showing the inability of NRG1 to rescue phosphorylation of ErbB3 and AKT resulting in PARP cleavage and Poziotinib-induced apoptosis in BCBM cells.

Figure 9.. Poziotinib effectively reduced brain metastases in mice

BCBM94 and BT474 cells were orthotopically xenografted into SCID and RAG2yc−/− mice. Upon detection of sizable brain metastases with MRI, the animals were treated with either Lapatinib (80mg/kg), Poziotinib (4mg/kg), or solvent control for two 5-day cycles with 2 days off treatment in between. (A) Treatment with Poziotinib resulted in a significant reduction of BCBM94 and BT474 brain metastatic tumors. The ROI-based volumetry was performed on pre- and post-treatment sets of MRI scans (n=4/group). Bar charts present mean +/− SD, **p<0.01. Representative MR images are shown below the charts (A, B). (C) Poziotinib is more brain penetrable than Lapatinib. Drug concentrations were measured by UPLC-MS/MS analysis of plasma and brain tissues taken 1 hour after the last drug administration (n=4/group). (D) Poziotinib abrogated ErbB3 phosphorylation, inhibited proliferation, and induced apoptosis within BCBM94 brain metastasis. H&E, IHC, and TUNEL analysis of post-treatment mouse brain FFPE tissues is shown for solvent control, Lapatinib and Poziotinib treatment groups. The white dashed lines mark the margins of the metastatic lesion. ErbB3 phosphorylation and presence of Ki67+ nuclei were observed only in lesions of the solvent control and Lapatinib groups. TUNEL assay shows fragmented DNA in green color. Magnification: H&E 100x, Ki67 200x, TUNEL and pErbB3 400x.