Targeting interferon response genes sensitizes aromatase inhibitor resistant breast cancer cells to estrogen-induced cell death.

INTRODUCTION: Estrogen deprivation using aromatase inhibitors (AIs) is currently the standard of care for postmenopausal women with hormone receptor-positive breast cancer. Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. The mechanism by which resistance occurs is still not completely known, however, recent studies suggest that impaired/defective interferon signaling might play a role. In the present study, we assessed the functional role of IFITM1 and PLSCR1; two well-known interferon response genes in AI resistance. METHODS: Real-time PCR and Western blot analyses were used to assess mRNA and protein levels of IFITM1, PLSCR1, STAT1, STAT2, and IRF-7 in AI-resistant MCF-7:5C breast cancer cells and AI-sensitive MCF-7 and T47D cells. Immunohistochemistry (IHC) staining was performed on tissue microarrays consisting of normal breast tissues, primary breast tumors, and AI-resistant recurrence tumors. Enzyme-linked immunosorbent assay was used to quantitate intracellular IFNα level. Neutralizing antibody was used to block type 1 interferon receptor IFNAR1 signaling. Small interference RNA (siRNA) was used to knockdown IFITM1, PLSCR1, STAT1, STAT2, IRF-7, and IFNα expression. RESULTS: We found that IFITM1 and PLSCR1 were constitutively overexpressed in AI-resistant MCF-7:5C breast cancer cells and AI-resistant tumors and that siRNA knockdown of IFITM1 significantly inhibited the ability of the resistant cells to proliferate, migrate, and invade. Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells. Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells. Lastly, neutralizing antibody against IFNAR1/2 and knockdown of STAT1/STAT2 completely suppressed IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells, thus confirming the involvement of the canonical IFNα signaling pathway in driving the overexpression of IFITM1 and other interferon-stimulated genes (ISGs) in the resistant cells. CONCLUSION: Overall, these results demonstrate that constitutive overexpression of ISGs enhances the progression of AI-resistant breast cancer and that suppression of IFITM1 and other ISGs sensitizes AI-resistant cells to estrogen-induced cell death.

Loss of pigment epithelium-derived factor: a novel mechanism for the development of endocrine resistance in breast cancer.

INTRODUCTION: Despite the benefits of endocrine therapies such as tamoxifen and aromatase inhibitors in treating estrogen receptor (ER) alpha-positive breast cancer, many tumors eventually become resistant. The molecular mechanisms governing resistance remain largely unknown. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that displays broad anti-tumor activity based on dual targeting of the tumor microenvironment (anti-angiogenic action) and the tumor cells (direct anti-tumor action). Recent studies indicate that PEDF expression is significantly reduced in several tumor types, including breast cancer, and that its reduction is associated with disease progression and poor patient outcome. In the current study, we investigated the role of PEDF in the development of endocrine resistance in breast cancer. METHODS: PEDF mRNA and protein levels were measured in several endocrine-resistant breast cancer cell lines including MCF-7:5C, MCF-7:2A, and BT474 and in endocrine-sensitive cell lines MCF-7, T47D, and ZR-75-1 using real-time PCR and western blot analyses. Tissue microarray analysis and immunohistochemistry were used to assess the PEDF protein level in tamoxifen-resistant breast tumors versus primary tumors. Lentiviruses were used to stably express PEDF in endocrine-resistant breast cancer cell lines to determine their sensitivity to tamoxifen following PEDF re-expression. RESULTS: We found that PEDF mRNA and protein levels were dramatically reduced in endocrine-resistant MCF-7:5C, MCF-7:2A, and BT474 breast cancer cells compared with endocrine-sensitive MCF-7, T47D, and ZR-75-1 cells, and that loss of PEDF was associated with enhanced expression of pSer167ERα and the receptor tyrosine kinase rearranged during transfection (RET). Importantly, we found that silencing endogenous PEDF in tamoxifen-sensitive MCF-7 and T47D breast cancer cells conferred tamoxifen resistance whereas re-expression of PEDF in endocrine-resistant MCF-7:5C and MCF-7:2A cells restored their sensitivity to tamoxifen in vitro and in vivo through suppression of RET. Lastly, tissue microarray studies revealed that PEDF protein was reduced in ~52.4% of recurrence tumors (31 out of 59 samples) and loss of PEDF was associated with disease progression and poor patient outcome. CONCLUSION: Overall, these findings suggest that PEDF silencing might be a novel mechanism for the development of endocrine resistance in breast cancer and that PEDF expression might be a predictive marker of endocrine sensitivity.

Phenotypic and molecular characterisation of type 3 von Willebrand disease in a cohort of Indian patients.

Severe type 3 VWD (VWD3) is characterised by complete absence or presence of trace amounts of non-functional von Willebrand factor (VWF). The study was designed to evaluate the VWF mutations in VWD3 patients and characterise the breakpoints of two identified homozygous novel large deletions. Patients were diagnosed by conventional tests and VWF multimer analysis. Mutation screening was performed in 19 VWD3 patients by direct sequencing of VWF including flanking intronic sequence and multiplex ligation-dependent probe amplification (MLPA) analysis. Breakpoint characterisation of two identified novel large deletions was done using walking primers and long spanning PCR. A total of 21 different mutations including 15 (71.4%) novel ones were identified in 17 (89.5%) patients. Of these mutations, five (23.8%) were nonsense (p.R1659*, p.R1779*, p.R1853*, p.Q2470*, p.Q2520*), one was a putative splice site (p.M814I) and seven (33.3%) were deletions (p.L254fs*48, p.C849fs*60, p.L1871fs*6, p.E2720fs*24) including three novel large deletions of exon 14-15, 80,830bp (-41510_657+7928A*del) and 2,231bp [1534-2072T_c.1692G*del(p.512fs*terminus)] respectively. A patient carried gene conversion comprising of pseudogene harbouring mutations. The missense mutations (p.G19R, p.K355R, p.D437Y, p.C633R, p.M771V, p.G2044D, p.C2491R) appear to play a major role and were identified in seven (36.8%) patients. In conclusion, a high frequency of novel mutations suggests the high propensity of VWF for new mutations. Missense and deletion mutations found to be a common cause of VWD3 in cohort of Indian VWD3 patients. Breakpoints characterisation of two large deletions reveals the double strand break and non-homologous recombination as deletions mechanism.

Characterisation of mutations and molecular studies of type 2 von Willebrand disease.

Type 2 von Willebrand disease (VWD) is characterised by qualitative defects in von Willebrand factor (VWF). Exon 28 of the VWF gene is known to be a hot spot for type 2 VWD mutations. The goal of this study was to characterise the mutations in VWF exon 28 and understand the molecular basis of phenotypes through in vitro and in silico studies. Mutation screening was performed in 56 type 2 VWD patients through direct sequencing. Expression vectors for five mutations were transiently expressed in 293-EBNA cells to understand the mutations pathology. Furthermore, in silico structure analysis was performed for 13 missense mutations. A total of 16 including eight novel mutations were detected in 23 (41%) patients. Of these, 15 were missense (including seven V1439M, A1464P, M1495L, I1509V, R1527Q, N1635I and A1647D novel ones) and one was a novel gene conversion. Expression studies and characterisation of recombinant VWF suggested the loss of VWF function for mutants P1266Q, V1439M and N1635I and gain of function for mutant R1308C. No apparent defect was seen in mutant N1231S. In silico structure analysis suggested the probable gain or loss of hydrogen/van der Waals interactions in 10 mutant proteins. In conclusion, type 2A mutations and gene conversion were found to be a common cause of type 2 VWD. Expression studies suggest the mutations N1635I for type 2A(II), P1266Q and V1439M for type 2M, R1308C for type 2B VWD and N1231S as a non-causative variant. Moreover, in silico studies of the mutants show the probable cause of respective phenotypes.