Clinicopathologic significance of nuclear HER4 and phospho-YAP(S127) in human breast cancers and matching brain metastases

Abstract

Background: Human epidermal growth factor receptor-4 (HER4) and yes-associated protein-1 (YAP) are candidate therapeutic targets in oncology. YAP's transcriptional coactivation function is modulated by the HER4 intracellular domain (HER4-ICD) in vitro, but the clinical relevance of this has not been established. This study investigated the potential for targeting the HER4-YAP pathway in brain metastatic breast cancer.

Methods: We performed immuno-phenotypic profiling of pathway markers in a consecutive breast cancer series with 25 years of clinical follow up (n = 371), and patient-matched breast and metastatic brain tumours (n = 91; 30 pairs).

Results: Membrane localisation of phospho-HER4 [pHER4(Y¹¹⁶²)] was infrequent in primary breast cancer, but very frequent in brain metastases (5.9% versus 75% positive), where it was usually co-expressed with pHER3(Y¹²⁸⁹) (p < 0.05). The presence of YAP in tumour cell nuclei was associated directly with nuclear pERK5(T²¹⁸/Y²¹⁰) (p = 0.003). However, relationships with disease-specific survival depended on oestrogen receptor (ER) status. Nuclear pYAP(S¹²⁷) was associated with smaller, good prognostic ER+ breast tumours (log-rank hazard-ratio 0.53; p = 9.6E^-03), but larger, poor prognostic triple-negative cancers (log-rank hazard-ratio 2.78; p = 1.7E^-02), particularly when co-expressed with nuclear HER4-ICD (p = 0.02). This phenotype was associated with stemness and mitotic instability markers (vimentin, SOX9, ID1, SPAG5, TTK, geminin; p < 0.05). YAP expression in brain metastases was higher than matched primary tumours; specifically, nuclear pYAP(S¹²⁷) in ER-negative cases (p < 0.05). Nuclear YAP was detected in ~70% of ER-negative, HER4-activated brain metastases.

Discussion: Our findings suggest that the canonical-mechanism where Hippo pathway-mediated phosphorylation of YAP ostensibly excludes it from the nucleus is dysfunctional in breast cancer. The data are consistent with pYAP(S¹²⁷) having independent transcriptional functions, which may include transducing neuregulin signals in brain metastases. Consistent with mechanistic studies implicating it as an ER co-factor, nuclear pYAP(S¹²⁷) associations with breast cancer clinical outcomes were dependent on ER status.

Conclusion: Preclinical studies investigating HER4 and nuclear YAP combination therapy strategies are warranted.

Keywords: HER4; biomarkers; brain metastasis; breast cancer.

Figure 1.

Expression and activation of HER4 in early breast cancer. (A) Representative breast tumour cores stained for HER4. (B) Chi-square analysis of the proportions of nuclear and cytoplasmic HER4 staining in major breast cancer subtypes. (C) Chi-square analysis of the proportions of cases exhibiting compartment specific HER4 staining. (D) KM analysis of the relationships between HER4 compartment categories (blue and green) and BCSS. Pie charts indicate proportion of cohort in each category. (E) Representative tumour cores stained for pHER4(Y¹¹⁶²). (F) Chi-square analysis of the proportions of cases with pHER4(M+) across molecular subtypes (i) and proliferative status of HER2+ cases (ii). (G) Kaplan–Meier analysis of relationships between pHER4(M) staining and BCSS. BCSS, breast cancer-specific survival; BLBC, basal-like breast cancer; HER4, human epidermal growth factor receptor-4; LumA/B, luminal A/B; Ki67 %TC+, percentage of tumour cells positive for proliferation marker Ki67; KM, Kaplan–Meier; pHER4(M+), pHER4 membrane positivity.

Figure 2.

Expression and activation of HER4 in brain-metastatic breast cancers and brain metastases. (A) Chi-square analysis of cyto.HER4, nu.4ICD and m.pHER4 in major breast cancer subtypes. *p < 0.05; ***p < 0.001; ****p < 0.0001. (B) Changes in cyto.HER4, nu.4ICD and m.pHER4 in individual matched cases, separated according to major breast cancer subtypes. Chi-square p-values indicate significant differences before and after metastasis to the brain. (C) Tile plot showing overall numbers of br.MBC and BrM co-expressing pHER receptors (columns are individual tumours). (D) Representative IHC analysis of samples from a patient whose HER2+ breast cancer recurred in the brain 7 years after diagnosis and treatment with HER2-targeted therapy. This case exemplifies concomitant induction of pHER2, pHER3 and pHER4. BrM, brain metastases; br.MBC, brain metastatic breast cancers; cyto.HER4, cytoplasmic HER4; HER4, human epidermal growth factor receptor-4; IC-NST, invasive carcinoma, no special type; IHC, immunohistochemistry; LumA/B, luminal A/B; m.pHER4, membrane-pHER4; nu.4ICD, nuclear HER4-ICD.

Figure 3.

Expression and activation of YAP in early breast cancer, and relationship with patient survival. (A) Representative images of breast cancer cores stained with antibodies specific for YAP and pYAP(S¹²⁷). (B) Contingency analysis in ER+ and TNBCs showing: (i) cyto.YAP intensity relative to nu.YAP intensity; (ii) cyto.pYAP(S¹²⁷) status relative to total cyto.YAP; (iii/iv) nu.pYAP(S¹²⁷) status relative to overall nuclear YAP levels or cyto.pYAP(S¹²⁷). Chi-square p-values shown. (C) KM analysis of relationships between YAP phosphorylation and BCSS in ER+ (left panel) and TNBC (right panel) cases. Analyses were designed to examine the effect of phosphorylation of the cytoplasmic or nuclear protein pools, and the relationships between pYAP(S¹²⁷) subcellular distribution and BCSS. Pie charts show relative proportions of cases in each category. Statistical significance determined using log rank tests. TNBC subgroups ‘nucleus>>cyto’ and ‘uniformly high’ were combined for statistical testing (bottom right). (D) (i) Relationship between YAP1 gene copy number and RNA expression. Kruskal–Wallis p-value shown. (ii) Proportions of TN and non-TN TCGA breast cancers with copy-number alterations. Chi-square p-value shown. (iii) KM analysis comparing survival of TNBCs (TCGA) with loss versus gain/amplification (amp) of the YAP1 gene. BCSS, breast cancer-specific survival; cyto.YAP, cytoplasmic YAP; ER+, oestrogen receptor positive; KM, Kaplan–Meier; nu.YAP, nuclear YAP; TCGA, The Cancer Genome Atlas; TN, triple negative; TNBC, triple negative breast cancer; YAP, yes-associated protein-1.

Figure 4.

Clinico-pathologic correlates of cytoplasmic- or nuclear-localised pYAP(S¹²⁷). Bar graphs show YAP phosphorylation status in the cytoplasm (i) or nucleus (ii) in ER+ and ER-negative cases; (iii) shows the overall distribution of pYAP(S¹²⁷). (A,B) Relationships between subcellular YAP phosphorylation and nu.pERK5. (C) Relationships between YAP and nu.4ICD in ER+ and ER-negative cases. Chi-square tests were used to determine statistical significance. (D) KM analysis (with log-rank tests) of the relationships between nu.pYAP(S¹²⁷), nu.4ICD and BCSS in ER+ and TNBC cases. (E) Proportions of TNBCs positive and negative for stemness and CIN markers according to nuclear YAP expression and phosphorylation. Chi-square p-value shown (*p = 0.05–0.01; **p < 0.01; p < 0.001). BCSS, breast cancer-specific survival; CIN, chromosomal instability; CK, cytokeratin; ER, oestrogen receptor; Gem, geminin; KM, Kaplan–Meier; nu.4ICD, nuclear HER4-ICD; nu.pERK5, nuclear pERK5; TNBC, triple negative breast cancer; Vim, vimentin; YAP, yes-associated protein-1.

Figure 5.

Expression and phosphorylation of YAP in breast cancers and matching brain metastases. (A) Levels and phosphorylation of the cytoplasmic and nuclear YAP pools (i/ii), and pYAP(S¹²⁷) distribution (iii) in a brain metastasis progression series comprised of the general breast cancer series (BC) plus matched br.MBC and BrM. (B) Changes in the proportions of BC, br.MBC and BrM expressing membrane-associated pHER4 and/or nuclear YAP (phosphorylated or unphosphorylated forms). Chi-square tests used to determine statistical significance (*p = 0.05–0.01; **p < 0.01; ***p < 0.001; ****p < 0.0001). BC, breast cancer series; BrM, brain metastases; br.MBC, brain-metastatic breast cancers; HER4, human epidermal growth factor receptor-4; YAP, yes-associated protein-1.