Hernandez, Brenda Y.

Abstract Sphingosine kinase 1 (SphK1) phosphorylates pro-apoptotic sphingosine to form pro-survival sphingosine-1-phosphate (S1P), leading to cancer progression. In breast cancer, high levels of SphK1 mRNA are found in 80% of the patients, with average increase of 2-fold in breast tumors compared with normal tissue from the same patient. Recent studies suggest that SphK1 expression is associated with disease-free survival in ER-negative and HER2-positive breast tumors. In addition, inhibition of SphK1 in HER2-positive breast cancer cell line reduced S1P-induced HER2 and ERK1/2 activation. While these evidences suggest that SphK1 may play an important role in HER2-positive breast cancer, the exact role of SphK1 in breast tumorigenesis remains unclear. The aim of this project is to define the role of SphK1 in HER2-positive breast tumorigenesis and to investigate whether the pathway components are potential target for breast cancer prevention and therapies. Here, we report that SphK1 is required for HER2/neu-induced breast tumorigenesis and SphK1 regulates claudin-2 (CLDN2) to promote tumor development perhaps by mediating HER2. In MMTV-neu transgenic mouse model, genetic deletion of SphK1, which was accompanied by reduced S1P and increased C16-ceramide levels, significantly inhibited breast tumor development, with reduced incidence (SphK1-/-, 19%; SphK1+/-, 26%; and SphK1+/+, 57%; p < 0.05) and multiplicity (SphK1-/-, 0.27 ± 0.13; SphK1+/-, 0.32 ± 0.07; and SphK1+/+, 0.67 ± 0.14 tumors per animal; p < 0.05). Unexpectedly, SphK1 deletion did not affect tumor growth as results showed no differences in tumor volume. However, this is not surprising when considering the fact that the tumors were allowed to grow until tumor reached 20 mm in longer diameter, at which they were sacrificed regardless of age. Interestingly, whole genome expression analysis by Illumina MouseWG-6 v2.0 Expression BeadChip Array revealed significant reduction of CLDN2 expression in tumors from SphK1 deficient mice (p < 0.0001). In support to these findings, overexpression of SphK1 in MDA-MB-453 human breast cancer cells, which HER2 is amplified but lacks hormone receptors, lead to increase in CLDN2 expression (p < 0.05). Furthermore, overexpression of SphK1 in MDA-MB-453 cells also enhanced cell proliferation, colony formation, and migration/invasion, and drug resistance to cisplatin. The relevance of this finings in humans was confirmed by immunostaining of SphK1 and CLDN2 in HER2-positive breast cancer tumors and normal tissues (n = 93 and 35 subjects, respectively). The results showed that the expression of SphK1 was higher in tumors compared to adjacent normal tissue (p < 0.0001). Furthermore, higher SphK1 expression was observed in high grade tumors (vs low grade; p < 0.001) and ER-PR- tumors (vs ER+PR+; p < 0.0001). Interestingly, we observed similar staining pattern of CLDN2 proteins, where CLDN2 expression was high in tumors (p < 0.0001), particularly in high grade tumors (p < 0.01) and ER-PR- tumors (p < 0.0001). Moreover, we found a strong correlation between SphK1 and CLDN2 expressions in tumors but not in normal tissues, confirming the results obtained in animal study. Taken together, these findings suggest that SphK1 may play a pivotal role in HER2-positive breast carcinogenesis, and targeting SphK1 and/or CLDN2 may represent a novel approach for HER2-positive breast cancer chemoprevention and/or treatment. Citation Format: Yoshiko Shimizu, Hideki Furuya, Paulette M. Tamashiro, Steve Goodison, Owen Chan, Ian Pagano, Kayoko Iino, Rafael Peres, Kanani Hokutan, Lenora W.M. Loo, Brenda Hernandez, Aung Naing, Charles J. Rosser, Toshihiko Kawamori. Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in MMTV-neu transgenic model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B17.

Abstract Genome-wide association studies (GWAS) of 10 different cancers have identified pleiotropic cancer predisposition loci across a region of chromosome 5p15.33 that includes the TERT and CLPTM1L genes. Of these, susceptibility alleles for pancreatic cancer have mapped to the CLPTM1L gene, thus prompting an investigation of the function of CLPTM1L in the pancreas. Immunofluorescence analysis indicated that CLPTM1L localized to the endoplasmic reticulum where it is likely embedded in the membrane, in accord with multiple predicted transmembrane domains. Overexpression of CLPTM1L enhanced growth of pancreatic cancer cells in vitro (1.3–1.5–fold; PDAY7 < 0.003) and in vivo (3.46-fold; PDAY68 = 0.039), suggesting a role in tumor growth; this effect was abrogated by deletion of two hydrophilic domains. Affinity purification followed by mass spectrometry identified an interaction between CLPTM1L and non-muscle myosin II (NMM-II), a protein involved in maintaining cell shape, migration, and cytokinesis. The two proteins colocalized in the cytoplasm and, after treatment with a DNA-damaging agent, at the centrosomes. Overexpression of CLPTM1L and depletion of NMM-II induced aneuploidy, indicating that CLPTM1L may interfere with normal NMM-II function in regulating cytokinesis. Immunohistochemical analysis revealed enhanced staining of CLPTM1L in human pancreatic ductal adenocarcinoma (n = 378) as compared with normal pancreatic tissue samples (n = 17; P = 1.7 × 10−4). Our results suggest that CLPTM1L functions as a growth-promoting gene in the pancreas and that overexpression may lead to an abrogation of normal cytokinesis, indicating that it should be considered as a plausible candidate gene that could explain the effect of pancreatic cancer susceptibility alleles on chr5p15.33. Cancer Res; 74(10); 2785–95. ©2014 AACR.