Mapping molecular subtype specific alterations in breast cancer brain metastases identifies clinically relevant vulnerabilities

The molecular events and transcriptional plasticity driving brain metastasis in clinically relevant breast tumor subtypes has not been determined. Here we comprehensively dissect genomic, transcriptomic and clinical data in patient-matched longitudinal tumor samples, and unravel distinct transcriptional programs enriched in brain metastasis. We report on subtype specific hub genes and functional processes, central to disease-affected networks in brain metastasis. Importantly, in luminal brain metastases we identify homologous recombination deficiency operative in transcriptomic and genomic data with recurrent breast mutational signatures A, F and K, associated with mismatch repair defects, TP53 mutations and homologous recombination deficiency (HRD) respectively. Utilizing PARP inhibition in patient-derived brain metastatic tumor explants we functionally validate HRD as a key vulnerability. Here, we demonstrate a functionally relevant HRD evident at genomic and transcriptomic levels pointing to genomic instability in breast cancer brain metastasis which is of potential translational significance

GLI1 promotes cell proliferation, viability, migration, invasion and colony formation of HCC cells.

<p>(A) Overexpression of GLI1 increases BrdU incorporation of Huh7 cells and silencing GLI1 expression in SNU398 cells decreases BrdU incorporation. (B) As assessed by the MTT assay, cell viability is increased at 48, 72 and 96 hours by overexpression of GLI1 in Huh7 cells and decreased at 24, 48, 72 and 96 hours by knockdown of GLI1 in SNU398 cells. (C) As assessed by the wound-healing assay, at both 24 and 48 hours, cell migration rate is increased in Huh7 cells by overexpression of GLI1 and decreased in SNU398 cells by silencing GLI1 expression. (D) Cell invasion is increased by overexpression of GLI1 in Huh7 cells and decreased by knockdown of GLI1 in SNU398 cells. (E) Overexpression of GLI1 promotes colony formation of Huh7 cells; in contrast, knockdown of GLI1 represses colony formation of SNU398 cells.</p

Knockdown of SNAI1 reverses the EMT phenotype induced by GLI1 in HCC cells.

<p>(A) SNAI1 siRNA decreases SNAI1 expression in Huh7 cells transfected stably with GLI1 expressing plasmid. SNAI1 siRNA increases E-cadherin expression (B) and decreases the expression of both N-cadherin (C) and Vimentin (D) in Huh7 cells transfected stably with GLI1 expressing plasmid.</p

Immunofluorescence studies confirm the role of GLI1 in the regulation of EMT in HCC cells.

<p>(A) As assessed by immunofluorescence staining and confocal microscopy, overexpression of GLI1 decreases E-cadherin protein expression and increases the protein expression of both N-cadherin and Vimentin in Huh7 cells. (B) As assessed by immunofluorescence staining and confocal microscopy, knockdown of GLI1 (GLI1 ASO) increases E-cadherin protein expression and decreases the protein expression of both N-cadherin and Vimentin in SNU398 cells. DAPI was used as nuclear staining.</p

GLI1 expression is positively associated with HCC recurrence.

<p>(A) Tumor recurrence after surgical resection in 139 patients with HCCs. Differences between the Kaplan-Meier curves of patients in the GLI1 high expresser group and those in GLI1 low/non expresser group using the log rank test. (B) Tumor recurrence after surgical resection in the 35 patients from the high GLI1 group and in the 35 patients from the low GLI1 group. Increased GLI1 expression in HCC tissues was significantly correlated with more rapid tumor recurrence after surgical resection of the primary tumor.</p

GLI1 is induced by TGFβ1 and mediates the mechanism by which TGFβ1 induces EMT in HCC cells.

<p>(A) TGFβ1 increases GLI1 expression in Huh7 cells. And TGFβ1 is also found to decrease E-cadherin expression, while increasing the expression of N-cadherin, Vimentin and SNAI1 in Huh7 cells. (B) Suppressing the TGFβ1 induced up-regulation of GLI1 in Huh7 cells by GLI1 ASO decreased the expression of both GLI1 and SNAI1 in Huh7 cells. (C) Suppressing the TGFβ1 induced up-regulation of GLI1 in Huh7 cells by GLI1 ASO increases E-cadherin expression and decreases the expression of both N-cadherin and Vimentin.</p