Immune-checkpoint blockade (ICB) combined with neoadjuvant chemotherapy improves pathological complete response in breast cancer (BC). To understand why only a subset of tumors respond to ICB, patients with hormone receptor-positive or triple-negative BC were treated with anti-PD1 prior to surgery. Paired pre-versus on-treatment biopsies from treatment-naïve patients receiving anti-PD-1 (n=29) or patients receiving neoadjuvant chemotherapy prior to anti-PD1 (n=11) were subjected to single-cell transcriptome, T-cell receptor and proteome profiling. One-third of tumors contained PD1-expressing T-cells, which clonally expanded upon anti-PD1 treatment irrespective of tumor subtype. Expansion mainly involved CD8 + T-cells with pronounced expression of cytotoxic-activity (PRF1, GZMB), immune-cell homing (CXCL13) and exhaustion markers (HAVCR2, LAG3), and CD4 + T-cells characterized by expression of T-helper-1 (IFNG) and follicular-helper (BCL6, CXCR5) markers. In pre-treatment biopsies, the relative frequency of immunoregulatory dendritic cells (PD-L1), specific macrophage phenotypes (CCR2 or MMP9) and cancer cells exhibiting MHC class I/II expression correlated positively with T-cell expansion. Conversely, undifferentiated preeffector/memory T-cells (TCF7, GZMK) or inhibitory macrophages (CXCR3, C3) were inversely correlated. Collectively, our data identify various immunophenotypes and associated gene sets that are positively or negatively correlated with T-cell expansion following anti-PD1. We shed light on the heterogeneity in treatment response to anti-PD1 in breast cancer.
Paracrine NRG1/HER3 signals initiated by BM-MSC and T-MC promote CRC cell progression, and high tNRG1 expression is associated with poor prognosis in CRC.
Increasing evidence supports the critical roles played by adipose tissue in breast cancer progression. Yet, the mediators and mechanisms are poorly understood. Here, we show that breast cancer-associated adipose tissue from freshly isolated tumors promotes F-actin remodeling, cellular scattering, invasiveness, and spheroid reorganization of cultured breast cancer cells. A combination of techniques, including transcriptomics, proteomics, and kinomics enabled us to identify paracrine secretion of oncostatin M (OSM) by cancer-associated adipose tissue. Specifically, OSM, expressed by CD45 þ leucocytes in the stromal vascular fraction, induced phosphorylation of STAT3 (pSTAT3-)Y705 and S727 in breast cancer cells and transcription of several STAT3-dependent genes, including S100 family members S100A7, S100A8, and S100A9. Autocrine activation of STAT3 in MCF-7 cells ectopically expressing OSMinduced cellular scattering and peritumoral neovascularization of orthotopic xenografts. Conversely, selective inhibition of OSM by neutralizing antibody and Jak family kinases by tofacitinib inhibited STAT3 signaling, peritumoral angiogenesis, and cellular scattering. Importantly, nuclear staining of pSTAT3-Y705 identified at the tumor invasion front in ductal breast carcinomas correlates with increased lymphovascular invasion. Our work reveals the potential of novel therapeutic strategies targeting the OSM and STAT3 axis in patients with breast cancer harboring nuclear pSTAT3-Y705. Cancer Res; 74(23);
CCN5 is a member of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family and was identified as an estrogen-inducible gene in estrogen receptor-positive cell lines. However, the role of CCN5 in breast carcinogenesis remains unclear. We report here that the CCN5 protein is localized mostly in the cytoplasm and in part in the nucleus of human tumor breast tissue. Using a heterologous transcription assay, we demonstrate that CCN5 can act as a transcriptional repressor presumably through association with histone deacetylase 1 (HDAC1). Microarray gene expression analysis showed that CCN5 represses expression of genes associated with epithelial-mesenchymal transition (EMT) as well as expression of key components of the transforming growth factor  (TGF-) signaling pathway, prominent among them TGF-RII receptor. We show that CCN5 is recruited to the TGF-RII promoter, thereby providing a mechanism by which CCN5 restricts transcription of the TGF-RII gene. Consistent with this finding, CCN5, we found, functions to suppress TGF--induced transcriptional responses and invasion that is concomitant with EMT. Thus, our data uncovered CCN5 as a novel transcriptional repressor that plays an important role in regulating tumor progression functioning, at least in part, by inhibiting the expression of genes involved in the TGF- signaling cascade that is known to promote EMT.CCN5 (previously known as WISP-2) is a 29-kDa protein member of the connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family (2, 5, 23). The CCN family is composed of six members grouped on the basis of similar structural analogies (7). CCN proteins appear to play important roles in several biological processes, including cell growth, adhesion, and migration as well as numerous endocrine-regulated functions (9,39,41). CCN proteins encompass four structural domains: an insulin-like growth factor-binding protein (IGF-BP) domain, a von Willebrand factor type C (VWC) domain, a thrombospondin (TSP-1) domain, and a cysteine knot (CT) domain reported to act as a potential proliferation module (29). Although the physiological function of CCN5 is not well defined, its domain structure suggests that its function may be different from that of other members of the CCN family. CCN5 contains only three structural domains and lacks the CT domain (7,8,35). It has been shown that CCN5 suppresses proliferation and its expression is reduced in cancers (14,31,35). Previously, we showed that upon hormone binding, the estrogen receptor (ER) directly regulates the ccn5 gene in all ER-positive breast cancer cell lines tested (17). Moreover, we found that CCN5 knockdown not only induced estradiol-independent growth of these cells, owing to a loss of estrogen receptor ␣ (ER␣) expression, but also promoted epithelial-mesenchymal transition (EMT) (18), a process involved in tumor invasiveness and metastasis (13,43,46,51).Consistent with its role in tumor progression, our recent studies have shown that CCN5 is ...
IntroductionAberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit.MethodsWe raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading.ResultsWith G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS).ConclusionsCapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.
Ductal carcinoma in situ (DCIS) is a heterogeneous malignant condition of the breast with an excellent prognosis. Until recently mastectomy was the standard treatment. As the results of the National Surgical Adjuvant Breast and Bowel Project-17 trial and the introduction of the Van Nuys Prognostic Index (VNPI) less radical therapies are used. Objectives are to identify clinicopathologic and biologic factors that may predict outcome. Cases of DCIS diagnosed in two Belgian University Centers were included. Paraffin-embedded material and Hematoxylin and Eosin stained slides of DCIS cases were reviewed and tumor size, margin width, nuclear grade, and comedo necrosis were assessed. Molecular markers (estrogen receptor, progesterone receptor, HER1-4, Ki67, and c-myc) were assayed immunohistochemically. Applied treatment strategies were correlated with the prospective use of the VNPI score. Kaplan-Meier survival plots were generated with log-rank significance and multiple regression analysis was carried out using Cox proportional hazards regression analysis; 159 patients were included with a median age of 54 years (range 29-78); 141 had DCIS and 18 DCIS with microinvasion. The median time of follow-up was 54 months (range 5-253). Twenty-three patients developed a recurrence (14.5%). The median time to recurrence was 46 months (range 5-253). Before the introduction of the VNPI, 37.5% of the DCIS patients showed a recurrence while thereafter 6.7% recurred (p < 0.005). Two recurrences occurred in the VNPI group I (7.1%); seven in the VNPI group II (8.5%) (median time to recurrence 66.3 months) and 14 in the VNPI group III (28.5%) (median time to recurrence 40.2 months) (disease-free survival [DFS]: p < 0.05). A Cox proportional hazards regression analysis indicated that tumor size, margin width, pathologic class, and age were independent predictors of recurrence, but none of the studied molecular markers showed this. Overexpression of HER4 in the presence of HER3 was found to be associated with a better DFS (p < 0.05). This study confirms the value of the VNPI score and questions the benefit of an aggressive approach in the low-risk DCIS lesions. Independent predictors for recurrence included size, margin width, pathologic class, and age, but none of the molecular markers were part of it. Overexpression of HER4 in the presence of HER3 was associated with a better DFS.
The secretory Rab27B small GTPase promotes invasive growth and metastasis in estrogen receptor (ER) a-positive breast cancer cells by orchestrating the peripheral targeting of vesicles secreting proinvasive growth regulators. Increased Rab27B expression is associated with poor prognosis in breast cancer patients. The molecular mechanisms of peripheral Rab27B secretory vesicle distribution are poorly understood. Mass spectrometry analysis on green fluorescent protein (GFP)-Rab27B vesicles prepared from GFP-Rab27B transfected MCF-7 human breast cancer cells detected eight subunits of the vacuolar H(1)-ATPase (V-ATPase) and the presence of V 0 a1 and V 0 d1 subunits was confirmed by Western blot analysis. Reversible inhibition of V-ATPase activity by bafilomycin A1 or transient silencing of V 0 a1 or V 0 d1 subunits demonstrated that V-ATPase controls peripheral localization and size of Rab27B vesicles. V-ATPase expression and activity further controls Rab27B-induced collagen type I invasion, cell-cycle progression and invasive growth in the chorioallantoic membrane assay. In agreement, Rab27B-dependent extracellular heat shock protein90a release and matrix metalloprotease-2 activation is markedly reduced by bafilomycin A1 and transient silencing of V 0 a1 and V 0 d1 subunits. Poor prognosis ERa-positive primary breast tumors expressing high levels of Rab27B also expressed multiple V-ATPase subunits and showed a strong cytoplasmic and peripheral V-ATPase V 1 E expression. In conclusion, inhibiting V-ATPase activity by interfering agents and drugs might be an effective strategy for blocking Rab27B-dependent proinvasive secretory vesicle trafficking in ERa-positive breast cancer patients.
Periductal myxoid stroma and reduced periductal decorin expression seem to be prognostic for overall ipsilateral locoregional recurrence in DCIS, whereas HER2 expression might be a more specific biomarker for invasive recurrence.
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