Estrogen receptor (ER) positive breast cancer (BC), the most abundant BC subtype, is notorious for poor response to neoadjuvant chemotherapy (NAC). The androgen receptor (AR) was reported to support ...estradiol-mediated ER activity in an in vitro system. Recently, ER-positive BC with fewer tumor infiltrating lymphocytes (TILs) was shown to have a better prognosis, opposite to the trend seen with ER-negative BC. We hypothesized that ER-positive BC with high expression of AR will have fewer TILs and an inferior response to NAC, but with a better prognosis. In both TCGA and METABRIC cohorts, AR expression was significantly higher in ER-positive BCs compared to ER-negatives (
< 0.001,
< 0.001, respectively) and it correlated with ER expression (
= 0.630,
= 0.509, respectively). In ER-positive tumors, AR high tumors enriched UV response down (NES = 2.01,
< 0.001), and AR low tumors enriched DNA repair (NES = -2.02,
< 0.001). AR high tumors were significantly associated with procancer regulatory T-cells, and AR low tumors were associated with anticancer immune cells, such as CD4, CD8, and Gamma-Delta T-cells and memory B-cells in ER-positive BC (
< 0.01). Further, cytolytic activity was significantly lower in AR high BC in both cohorts. Finally, AR high tumors had a significantly lower rate of attaining pathological complete response to NAC (GSE22358), but better survival. In conclusion, our results demonstrated that high AR has fewer tumor infiltrating lymphocytes as well as cytolytic activity and an inferior response to NAC, but better survival in ER-positive BC.
Annexin A1 (ANXA1) is a phospholipid-linked protein involved in inflammation, immune response, and mast cell reactivity. Recently, we reported that ANXA1 is associated with aggressive features of ...triple-negative breast cancer (TNBC); however, its clinical relevance remains controversial. We hypothesized that human TNBC with high expression of ANXA1 mRNA is associated with pro-cancerous immune cell infiltration, including mast cells, and with an aggressive phenotype. Clinical and RNA-seq data were obtained from The Cancer Genome Atlas (TCGA,
= 1079) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (
= 1904). TNBC patients had significantly higher levels of ANXA1 expression compared to the other subtypes in both TCGA and METABRIC cohorts (
< 0.001). ANXA1 protein expression was assessed by immunohistochemistry in Japanese TNBC patient cohort (
= 48), where 17 cases (35.4%) had positive ANXA1 staining, and their overall survival was significantly shorter compared with negative staining group (
= 0.008). The CIBERSORT algorithm was used to calculate immune cell infiltrations. ANXA1 high tumors were associated with activated mast cells and M2 macrophages (
> 0.01), but did not show any association with tumor heterogeneity nor cytolytic activity. High expression of ANXA1 group enriched inflammation, epithelial-to-mesenchymal transition (EMT), and angiogenesis-related genes in a gene set enrichment assay in both cohorts. To our knowledge, this is the first study to demonstrate that ANXA1 is associated with infiltration of mast cells and inflammation that is associated with the aggressive phenotype of TNBC, such as EMT and angiogenesis.
Sphingosine kinase 2 (SphK2) is known to phosphorylate the nuclear sphingolipid metabolite to generate sphingosine‐1‐phosphate (S1P). Nuclear S1P is involved in epigenetic regulation of gene ...expression; however, the underlying mechanisms are not well understood. In this work, we have identified the role of nuclear S1P and SphK2 in regulating hypoxia‐responsive master transcription factors hypoxia‐inducible factor (HIF)‐1α/2α, and their functions in breast cancer, with a focus on triple‐negative breast cancer (TNBC). We have shown SphK2 is associated with HIF‐1α in protein complexes, and is enriched at the promoters of HIF target genes, including vascular endothelial growth factor (VEGF), where it enhances local histone H3 acetylation and transcription. S1P specifically binds to the PAS domains of HIF‐1α. SphK2, and HIF‐1α expression levels are elevated in metastatic estrogen receptor‐positive (ER+) and TNBC clinical tissue specimens compared to healthy breast tissue samples. To determine if S1P formation in the nucleus by SphK2 is a key regulator of HIF functions, we found using a preclinical TNBC xenograft mouse model, and an existing selective SphK2 inhibitor K‐145, that nuclear S1P, histone acetylation, HIF‐1α expression, and TNBC tumor growth were all reduced in vivo. Our results suggest that S1P and SphK2 in the nucleus are linked to the regulation of HIF‐1α/2α functions associated with breast cancer progression, and may provide potential therapeutic targets.
Patient-Derived Xenograft (PDX) is now accepted as a murine model that better mimics human cancer when compared to a conventional cancer cell-line inoculation model. Some claim the advantage of ...orthotopic site implantation of patient tumor (OS) over ectopic implantation into the subcutaneous space (SQ); however, there has been no study that describes a head-to-head comparison of oncological differences between these two models to date. We hypothesize that OS tumors re-transplant and grow better than SQ tumors and are therefore a better model to evaluate tumor aggressiveness. Breast cancer PDXs were generated using the tumors derived from 11 patients into NOD scid gamma (NSG) mice. We used six ER(+)HER2(−) tumors and five triple negative (TN) tumors for a total of 11 tumors. Five PDX lines grew for an overall engraftment rate of 45%. We present our OS implantation method in detail. The re-transplantation rate of TN tumors in each transplant site was significantly higher in OS when compared to SQ tumors (70.1% vs. 32.1%,
p
< 0.01). OS tumors grow significantly faster than SQ tumors. Similarly, OS tumors demonstrated significantly more mitotic figures and Ki-67 positive cells than SQ tumors. The tumor re-transplantation rate significantly increased by the second and third generations with the OS method. The time from implantation to development of a palpable tumor dramatically decreased after the first passage. PDX of ER(+) tumors demonstrated significantly lower engraftment rates and slower tumor growth than TN tumors, which remarkably improved by the first passage. Orthotopically implanted PDX tumors showed better re-transplantation rates, greater tumor size, and more significant growth compared to the subcutaneously implanted model.
Tissue biopsy is the gold standard for diagnosis and morphological and immunohistochemical analyses to characterize cancer. However, tissue biopsy usually requires an invasive procedure, and it can ...be challenging depending on the condition of the patient and the location of the tumor. Even liquid biopsy analysis of body fluids such as blood, saliva, gastric juice, sweat, tears and cerebrospinal fluid may require invasive procedures to obtain samples. Liquid biopsy can be applied to circulating tumor cells (CTCs) or nucleic acids (NAs) in blood. Recently, urine has gained popularity due to its less invasive sampling, ability to easily repeat samples, and ability to follow tumor evolution in real-time, making it a powerful tool for diagnosis and treatment monitoring in cancer patients. With the development and advancements in extraction methods of urinary substances, urinary NAs have been found to be closely related to carcinogenesis, metastasis, and therapeutic response, not only in urological cancers but also in non-urological cancers. This review mainly highlights the components of urine liquid biopsy and their utility and limitations in oncology, especially in non-urological cancers.
Cell adhesion is indispensable for appropriate tissue architecture and function in multicellular organisms. Besides maintaining tissue integrity, cell adhesion molecules, including tight-junction ...proteins claudins (CLDNs), exhibit the signaling abilities to control a variety of physiological and pathological processes. However, it is still fragmentary how cell adhesion signaling accesses the nucleus and regulates gene expression.
By generating a number of knockout and rescued human breast cell lines and comparing their phenotypes, we determined whether and how CLDN4 affected breast cancer progression in vitro and in vivo. We also identified by RNA sequencing downstream genes whose expression was altered by CLDN4-adhesion signaling. Additionally, we analyzed by RT-qPCR the CLDN4-regulating genes by using a series of knockout and add-back cell lines. Moreover, by immunohistochemistry and semi-quantification, we verified the clinicopathological significance of CLDN4 and the nuclear receptor LXRβ (liver X receptor β) expression in breast cancer tissues from 187 patients.
We uncovered that the CLDN4-adhesion signaling accelerated breast cancer metabolism and progression via LXRβ. The second extracellular domain and the carboxy-terminal Y197 of CLDN4 were required to activate Src-family kinases (SFKs) and the downstream AKT in breast cancer cells to promote their proliferation. Knockout and rescue experiments revealed that the CLDN4 signaling targets the AKT phosphorylation site S432 in LXRβ, leading to enhanced cell proliferation, migration, and tumor growth, as well as cholesterol homeostasis and fatty acid metabolism, in breast cancer cells. In addition, RT-qPCR analysis showed the CLDN4-regulated genes are classified into at least six groups according to distinct LXRβ- and LXRβS432-dependence. Furthermore, among triple-negative breast cancer subjects, the "CLDN4-high/LXRβ-high" and "CLDN4-low and/or LXRβ-low" groups appeared to exhibit poor outcomes and relatively favorable prognoses, respectively.
The identification of this machinery highlights a link between cell adhesion and transcription factor signalings to promote metabolic and progressive processes of malignant tumors and possibly to coordinate diverse physiological and pathological events.
The vast majority of mortality in breast cancer results from distant metastasis. Brain metastases occur in as many as 30% of patients with advanced breast cancer, and the 1-year survival rate of ...these patients is around 20%. Pre-clinical animal models that reliably reflect the biology of breast cancer brain metastasis are needed to develop and test new treatments for this deadly condition. The patient-derived xenograft (PDX) model maintains many features of a donor tumor, such as intra-tumor heterogeneity, and permits the testing of individualized treatments. However, the establishment of orthotopic PDXs of brain metastasis is procedurally difficult. We have developed a method for generating such PDXs with high tumor engraftment and growth rates. Here, we describe this method and identify variables that affect its outcomes. We also compare the brain-orthotopic PDXs with ectopic PDXs grown in mammary pads of mice, and show that the responsiveness of PDXs to chemotherapeutic reagents can be dramatically affected by the site that they are in.
Due to the heterogeneous nature of breast cancer, metastasis organotropism has been poorly understood. This study assessed the specific cancer-related gene expression changes occurring with ...metastatic breast cancer recurrence to distant organs compared with non-metastatic breast cancer. We found that several secreted mediators encoding genes notably, LCN2 and S100A8 overexpressed at the distant metastatic site spine (LCN2, 5-fold; S100A8, 6-fold) and bone (LCN2, 5-fold; S100A8, 3-fold) vs. primary tumors in the syngeneic implantation/tumor-resection metastasis mouse model. In contrast, the ESM-1 encoding gene is overexpressed in the primary tumors and markedly downregulated at distant metastatic sites. Further digging into TCAGA-BRCA, SCAN-B, and METABRIC cohorts data analysis revealed that LCN2, S100A8, and ESM-1 mediators encoding individual gene expression scores were strongly associated with disease-specific survival (DSS) in the METABRIC cohort (hazard ratio (HR) > 1, p < 0.0004). The gene expression scores predicted worse clinically aggressive tumors, such as high Nottingham histological grade and advanced cancer staging. Higher gene expression score of ESM-1 gene was strongly associated with worse overall survival (OS) in the triple-negative breast cancer (TNBC) and hormonal receptor (HR)-positive/HER2-negative subtype in METABRIC cohort, HER2+ subtype in TCGA-BRCA and SCAN-B breast cancer cohorts. Our data suggested that mediators encoding genes with prognostic and predictive values may be clinically useful for breast cancer spine, bone, and lung metastasis, particularly in more aggressive subtypes such as TNBC and HER2+ breast cancer.
Annexin A1 (ANXA1) is a calcium-dependent phospholipid-linked protein, involved in anti-inflammatory effects, regulation of cellular differentiation, proliferation and apoptosis. While many studies ...have investigated the ANXA1 expression in various tumor types, the role of ANXA1 is not fully understood. Therefore, in the present study, we evaluated the ANXA1 expression in 211 breast cancer patients and compared the levels with clinicopathological factors. ANXA1 was positively expressed in 31 (14.7%) of the 211 cases in our cohort, and these positive cases were associated with triple-negative breast cancer (TNBC) (P=0.007) and venous invasion (P=0.028). The in vitro cell experiment found that the MDA-MB-231 cell line, which is a TNBC cell line, highly expressed ANXA1. Using this cell line, the functional role of ANXA1 in breast cancer was revealed and the knockdown of ANXA1 by specific siRNA demonstrated a significant reduction in cellular invasion. Further experiments indicated that ANXA1 was induced by hypoxia with hypoxia-inducible factor-1α induction. These results suggested that ANXA1, which enhanced breast cancer invasion and metastasis under hypoxia, were significantly associated with the worst patient outcome. This is particularly noted in TNBC, the group of breast cancer with the worst outcome for which new therapeutic implications are required.
Elderly patients are known to have a worse prognosis for breast cancer. This is commonly blamed on their medical comorbidities and access to care. However, in addition to these social issues, we ...hypothesized that the extreme elderly (octogenarians-patients over 80 years old) have biologically worse cancer with unfavorable tumor immune microenvironment. The Cancer Genomic Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were analyzed. The control (aged 40-65) and octogenarians numbered 668 and 53 in TCGA and 979 and 118 in METABRIC, respectively. Octogenarians had significantly worse breast cancer-specific survival in both cohorts (
< 0.01). Octogenarians had a higher ER-positive subtype rate than controls in both cohorts. Regarding PAM50 classification, luminal-A and -B subtypes were significantly higher in octogenarians, whereas basal and claudin-low subtypes were significantly lower (
< 0.05) in octogenarians. There was no difference in tumor mutation load, intratumor heterogeneity, or cytolytic activity by age. However, the octogenarian cohort was significantly associated with high infiltration of pro-cancer immune cells, M2 macrophage, and regulatory T cells in both cohorts (
< 0.05). Our results demonstrate that octogenarians' breast cancer is associated with worse survival and with an unfavorable tumor immune microenvironment.
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