Transcriptome-wide identification of mRNAs and lincRNAs associated with trastuzumab-resistance in HER2-positive breast cancer

Abstract

Approximately, 25-30% of early-stage breast tumors are classified at the molecular level as HER2-positive, which is an aggressive subtype of breast cancer. Amplification of the HER2 gene in these tumors results in a substantial increase in HER2 mRNA levels, and consequently, HER2 protein levels. HER2, a transmembrane receptor tyrosine kinase (RTK), is targeted therapeutically by a monoclonal antibody, trastuzumab (Tz), which has dramatically improved the prognosis of HER2-driven breast cancers. However, ~30% of patients develop resistance to trastuzumab and recur; and nearly all patients with advanced disease develop resistance over time and succumb to the disease. Mechanisms of trastuzumab resistance (TzR) are not well understood, although some studies suggest that growth factor signaling through other receptors may be responsible. However, these studies were based on cell culture models of the disease, and thus, it is not known which pathways are driving the resistance in vivo. Using an integrative transcriptomic approach of RNA isolated from trastuzumab-sensitive and trastuzumab-resistant HER2+ tumors, and isogenic cell culture models, we identified a small set of mRNAs and lincRNAs that are associated with trastuzumab-resistance (TzR). Functional analysis of a top candidate gene, S100P, demonstrated that inhibition of S100P results in reversing TzR. Mechanistically, S100P activates the RAS/MEK/MAPK pathway to compensate for HER2 inhibition by trastuzumab. Finally, we demonstrated that the upregulation of S100P appears to be driven by epigenomic changes at the enhancer level. Our current findings should pave the path toward new therapies for breast cancer patients.

Keywords: HER2; breast cancer; cancer therapy; drug resistance; trastuzumab-resistance.

Figure 1. Identification of differentially expressed genes in trastuzumab-resistant (TzR) vs trastuzumab-sensitive (TzS) tumors by RNA-seq

(A) A schematic outlining overall study design. Differentially expressed genes identified in TzS vs TzR human HER2+ tumors in vivo were intersected with differentially expressed genes identified in TzS vs TzR BT474 cell lines, which led to a small list of mRNAs and lincRNAs that are associated with TzR. (B) Heatmap representation of the differentially expressed mRNAs in TzS patients (responders) vs TzR patients (non-responders). (C) Pathway analysis of differentially expressed mRNAs in tumors in vivo. Pathway names are listed on the Y-axis, the percentage of genes affected in each pathway is indicated on the X-axis, and a p-value is given at the end of each bar.

Figure 2. Characterization of HER2-positive trastuzumab-sensitive (TzS) and trastuzumab-resistant (TzR) BT474 breast cancer cell lines

(A–B) Cell proliferation analysis of TzS and TzR BT474 cell lines with either (A) one dose of 10 μg/ml trastuzumab at 0 hr or (B) 10 μg/ml trastuzumab at 0 and 48 hours demonstrated that while TzS BT474 cells is significantly decreased by trastuzumab, TzR cells proliferation is not affected. (C) Western blot analysis demonstrates that both TzS and TzR BT474 cells have comparable levels of HER2 protein. (D) Trastuzumab (10 μg/ml) treatment significantly affects p-HER2 levels in TzS BT474 cells within 24 hours post treatment. (E) HER2 protein levels stay relatively constant in TzS BT474 cells with trastuzumab treatment over 96 hours. (F) p-HER2 levels decrease significantly at 24 hours post trastuzumab treatment, and remain undetectable at a 96 h timepoint. (G–H) Decreased levels of HER2 and p-HER2 levels are observed in TzR cells at 48 h post trastuzumab treatment.

Figure 3. Numerous mRNAs and lincRNAs are differentially expressed in TzS vs TzR BT474 cells

Heatmap representation of differentially expressed (A) mRNAs and (B) lincRNAs in TzS BT474 vs TzR BT474 isogenic cell lines.

Figure 4. Top candidate mRNAs associated with trastuzumab-resistant (TzR)

The expression of each mRNA in each tumor sample (blue dots: TzS, red dots: TzR) is shown as FPKM value based on RNA-seq analysis. HER2 mRNA levels are shown as a control and do not significantly change between TzS vs TzR tumors. By contrast, mRNAs identified in our analysis show striking upregulation in TzR tumors (red) vs the majority of TzS tumors (blue).

Figure 5. S100P is upregulated in trastuzumab-resistant cells

(A) S100P is highly upregualted in HER2+ tumors as compared to matched normal breast tissues (TCGA RNA-seq data). (B) S100P mRNA is elevated in two independently generated TzR vs TzS BT474 cells. (C) S100P protein is elevated in TzR vs TzS BT474 and SKBR3 cells as measured by western blot analysis.

Figure 6. Knock down of S100P partially reverses TzR in cell culture

Cell proliferation analysis using MTS colorimetric assay demonstrated that TzR BT474 cells treated with either mock vs trastuzumab (10 μg/ml) for 96 hours show decreased proliferation rate when S100P levels are depleted with two independent shRNAs targeting S100P vs a negative control shRNA targeting GFP.

Figure 7. S100P activates Ras/MEK/MAPK pathway

(A) BT474 cells infected with shS100P lentivirus or control shGFP lentivirus were plated into soft agar to assess anchorage independent growth (AIG). Cells were grown in the presence of 10 μg/mL Trastuzumab for 3 weeks. (*P < 0.01, Student's t test). (B) Trastuzumab treatment inhibits p-MAPK levels in TzS BT474 cells, but the addition of recombinant S100P protein is sufficient to restore p-MAPK levels. (C) Knock down of S100P in TzR cells results in significant decrease in p-MAPK levels when cells are treated with trastuzumab, further supporting a role of S100P in trastuzumab-resistance.

Figure 8. S100P upregulation in TzR cells is wired at the epigenetic level

All high confidence H3K4me1 and H3K27ac peaks (MACS, p-value < 1E-9) within 150KB of the TSS for top genes associated with trastuzumab-resistance were retrieved and plotted as log2 ratio of the ChIP signal in TzR BT474 vs TzS BT474 cell line. Red lines demarcate the median signal fold change for each gene. (top panel) Each data point corresponds to relative levels of H3K4me1 ChIP-seq signals (trastuzumab-resistant/ trastuzumab-sensitive) located within 150-kb of each of the 16 dysregulated genes. (bottom panel) Same as top, but for H3K27ac.