Both t-Darpp and DARPP-32 can cause resistance to trastuzumab in breast cancer cells and are frequently expressed in primary breast cancers

Abstract

The clinical use of trastuzumab (Herceptin), a humanized antibody against the HER2 growth factor receptor, has improved survival in patients with breast tumors with ERBB2 amplification and/or over-expression. However, most patients with advanced ERBB2 amplified breast cancers whose tumors initially respond to trastuzumab develop resistance to the drug, leading to tumor progression. To identify factors responsible for acquired resistance to trastuzumab, gene expression profiling was performed on subclones of an ERBB2 amplified breast cancer cell line, BT474, which had acquired resistance to trastuzumab. The most overexpressed gene in these subclones was PPP1R1B, encoding the DARPP-32 phosphatase inhibitor. Western analysis revealed that only the truncated isoform of the DARPP-32 protein, t-Darpp, was overexpressed in the trastuzumab resistant cells. Using gene silencing experiments, we confirmed that t-Darpp over-expression was required for trastuzumab resistance in these cells. Furthermore, transfecting t-Darpp in parental BT-474 cells conferred resistance to trastuzumab, suggesting that t-Darpp expression was sufficient for trastuzumab resistance. We also found that t-Darpp over-expression was associated with Akt activation and that the T75 residue in t-Darpp was required for both Akt activation and trastuzumab resistance. Finally, we found that full-length DARPP-32 and t-Darpp are expressed in a majority of primary breast tumors. Over-expression of full-length DARPP-32 can also confer resistance to trastuzumab and, moreover, is associated with a poor prognostic value in breast cancers. Thus, t-Darpp and DARPP-32 expression are novel prognostic and predictive biomarkers in breast cancer.

Helicobacter pylori-induced cell death is counteracted by NF-κB-mediated transcription of DARPP-32

“…3 Recent studies have shown that DARPP-32 promotes cancer cell survival, drug resistance and invasion. [5][6][7][8][9] However, the mechanisms that regulate DARPP-32 expression and promote gastric carcinogenesis remain unclear. Infection with Helicobacter pylori has been classified by the WHO as a group 1 carcinogen 10 and considered a major risk factor for the development of gastric cancer.…”

t-Darpp stimulates protein kinase A activity by forming a complex with its RI regulatory subunit

“…All of these results suggest that T39 phosphorylation plays an important role in t-Darpp’s capacity to modulate PKA activity. This same site has already been shown to be critical for t-Darpp’s ability to activate EGFR signaling [21], to cause sustained Akt phosphorylation and to confer trastuzumab resistance [17], perhaps suggesting that these outcomes are downstream effects of t-Darpp-mediated PKA stimulation.…”

“…Darpp-32 inhibits PP1 via a protein-protein interaction that requires the N-terminal domain that is absent from t-Darpp, but the molecular mechanism by which Darpp-32 inhibits PKA is not known. We focused on the T39 phosphorylation site in t-Darpp because phosphorylation at this site is required for t-Darpp’s ability to increase trastuzumab resistance and PI3K/Akt signaling [17,21] and phosphorylation at the analogous T75 site in Darpp-32 converts Darpp-32 from a PP1 inhibitor into a PKA inhibitor [20]. …”

“…Phosphorylation at the T75 site in Darpp-32 converts it into a PKA inhibitor [20], whereas phosphorylation at T39, the analogous site in t-Darpp (see Fig. 1), is required for trastuzumab resistance and for activation of the PI3K/Akt pathway [17,21]. The role for T39 phosphorylation in t-Darpp-mediated PKA activation has not been previously reported.…”

Role of trastuzumab in the management of HER2-positive metastatic breast cancer

“…Overexpression of both DARPP32 and t-DARPP led to increased phosphorylation of Akt and increased BCL2 protein levels in trastuzumab-resistant cell lines 79 and in vivo. 80 Moreover, t-DARPP contributes to trastuzumab resistance by blocking Abbreviations: HER2, human epidermal growth factor receptor 2; NK, natural killer; FcγR, fragment crystallizable region gamma receptor; Src, Rous sarcoma tyrosine kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphatidylinositol 3-kinases; Akt, serine/threonine protein kinase; mTor, mammalian target of rapamycin; DARPP-32, dopamine-and cyclic-AMP-regulated phosphoprotein; t-DARPP, truncated form of dopamine-and cyclic-AMP-regulated phosphoprotein; MMP9, matrix metallopeptidase 9; p95, truncated form of HER2; Muc4, mucin 4; IGF-1R, insulin-like growth factor receptor 1; Met, hepatocyte growth factor receptor. …”