Abstract LB-315: Overexpression of prolactin inducible protein (PIP) in 4T1 cells leads to delayed tumor onset and reduced tumor size in experimental model of mouse breast cancer
Authors: Edechi Chidalu A. , Blanchard Anne , Kung Sam , Uzonna Jude , Myal Yvonne 
Published: August 2019

Abstract
Background: According to the WHO, breast cancer is the most common cancer among women, affecting over 2.1 million people annually. The prolactin inducible protein (PIP) was identified by our group as an abundantly secreted protein in some human breast cancer cell lines. Studies show that over 90% of breast cancers express PIP to varying degrees and PIP expression is associated with better prognosis and patient response to chemotherapy. However, a definitive role for PIP in breast cancer pathogenesis is not known. We previously found that in addition to its role in innate immunity, PIP plays a role in adaptive immune response because its deficiency was associated with defective type 1 T helper cell (Th1) activity, a critical immune component necessary for anti-tumor immunity. Here, we directly assessed the role of PIP in the pathogenesis of breast cancer by developing transplantable mouse models of breast cancer using PIP over-expressing 4T1 mouse breast cancer cell line.
Methods: Lentiviral transduction was used to generate PIP overexpressing 4T1 and its corresponding empty vector control. Fluorescent microscopy, flow cytometry and Western blot were used to confirm successful transduction and PIP expression. In vitro functional assays were performed to characterise this cell line, including XTT assay and cell counting to assess cell viability and proliferation; and scratch and Transwell migration assays to assess cell migration. The effect of PIP expression on sensitivity to doxorubicin, cisplatin, etoposide and tamoxifen was evaluated in vitro. To assess the impact of PIP expression on breast tumorigenesis in vivo, the cells were injected into syngeneic immunocompetent mice and tumor latency, size and progression were monitored. The immune phenotype and cytokine response were evaluated in the tumors, spleens and lymph nodes by flow cytometry.
Results: Western blot analysis using cell lysate and culture media confirmed PIP expression and secretion respectively. In vitro functional assays showed comparable rates of proliferation, migration and response to chemotherapeutic compounds in PIP overexpressing 4T1 compared to control. In vivo studies showed that overexpression of PIP leads to delayed tumor onset, smaller tumor size in the PIP group compared to the control group. PIP group showed increased frequency of NK cells and dendritic cells and reduced frequency of CD4+IL4+ T-cells in the tumor.
Conclusion: Collectively, these studies show that PIP overexpression had no significant effect on cancer cell proliferation, migration, and response to chemotherapeutic agents in vitro but affects the onset and progression of tumor in mice, suggesting that PIP does not act directly on breast cancer cells but may do so indirectly by altering the nature of immune response.
Citation Format: Chidalu A. Edechi, Anne Blanchard, Sam Kung, Jude Uzonna, Yvonne Myal. Overexpression of prolactin inducible protein (PIP) in 4T1 cells leads to delayed tumor onset and reduced tumor size in experimental model of mouse breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-315.