Escape In Breast Cancer Research Articles

GFPT1 accelerates immune escape in breast cancer by modifying PD-L1 via O-glycosylation

BackgroundImmune escape is one of the causes of poor prognosis in breast cancer (BC). Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first speed-limiting enzyme of the hexosamine biosynthesis pathway (HBP) and is essential for the progression of BC. Nevertheless, the mechanism of the influence of GFPT1 in BC immune escape is not clear.MethodsFirst, the level of GFPT1 in BC was analyzed by starbase, and GFPT1 expression in BC tissues was measured by qRT-PCR, western blot and IHC. Then, the O-GlcNAc levels were detected by western blot. Thereafter, Co-IP was applied to examine the relationship between GFPT1 and PD-L1. At last, a mouse model was constructed for validation in vivo.ResultsFirstly, we discovered that GFPT1 was obviously strengthened in BC. Knockdown or introduction of GFPT1 correspondingly degraded and elevated O-GlcNAc levels in cells. Further researches revealed that there was a reciprocal relationship between GFPT1 and PD-L1. Mechanistically, we disclosed that GFPT1 enhanced PD-L1 protein stability through O-glycosylation. More interestingly, GFPT1 accelerated BC cell immune escape via upregulation of O-glycosylation-modified PD-L1. In vivo, silencing of GFPT1 attenuated immune escape of BC cells by reducing PD-L1 levels.ConclusionGFPT1 promoted BC progression and immune escape via O-glycosylation-modified PD-L1. GFPT1 may be a potential target for BC therapy.

Soluble NKG2DLs Are Elevated in Breast Cancer Patients and Associate with Disease Outcome.

Ligands of the natural killer group 2D (NKG2DL) family are expressed on malignant cells and are usually absent from healthy tissues. Recognition of NKG2DLs such as MICA/B and ULBP1-3 by the activating immunoreceptor NKG2D, expressed by NK and cytotoxic T cells, stimulates anti-tumor immunity in breast cancer. Upregulation of membrane-bound NKG2DLs in breast cancer has been demonstrated by immunohistochemistry. Tumor cells release NKG2DLs via proteolytic cleavage as soluble (s)NKG2DLs, which allows for effective immune escape and is associated with poor prognosis. In this study, we collected serum from 140 breast cancer (BC) and 20 ductal carcinoma in situ (DCIS) patients at the time of initial diagnosis and 20 healthy volunteers (HVs). Serum levels of sNKG2DLs were quantified through the use of ELISA and correlated with clinical data. The analyzed sNKG2DLs were low to absent in HVs and significantly higher in BC patients. For some of the ligands analyzed, higher sNKG2DLs serum levels were associated with the classification of malignant tumor (TNM) stage and grading. Low sMICA serum levels were associated with significantly longer progression-free (PFS) and overall survival (OS). In conclusion, we provide the first insights into sNKG2DLs in BC patients and suggest their potential role in tumor immune escape in breast cancer. Furthermore, our observations suggest that serum sMICA levels may serve as a prognostic parameter in the patients analyzed in this study.

Converging and evolving immuno-genomic routes toward immune escape in breast cancer

The interactions between tumor and immune cells along the course of breast cancer progression remain largely unknown. Here, we extensively characterize multiple sequential and parallel multiregion tumor and blood specimens of an index patient and a cohort of metastatic triple-negative breast cancers. We demonstrate that a continuous increase in tumor genomic heterogeneity and distinct molecular clocks correlated with resistance to treatment, eventually allowing tumors to escape from immune control. TCR repertoire loses diversity over time, leading to convergent evolution as breast cancer progresses. Although mixed populations of effector memory and cytotoxic single T cells coexist in the peripheral blood, defects in the antigen presentation machinery coupled with subdued T cell recruitment into metastases are observed, indicating a potent immune avoidance microenvironment not compatible with an effective antitumor response in lethal metastatic disease. Our results demonstrate that the immune responses against cancer are not static, but rather follow dynamic processes that match cancer genomic progression, illustrating the complex nature of tumor and immune cell interactions.

Deubiquitinating Enzyme MINDY1 Facilitates Immune Escape in Breast Cancer by Maintaining the Stability of Immune Checkpoint Protein PD-L1.

Deubiquitinating Enzyme MINDY1 Facilitates Immune Escape in Breast Cancer by Maintaining the Stability of Immune Checkpoint Protein PD-L1.

Circular RNA hsa_circ_0067842 facilitates tumor metastasis and immune escape in breast cancer through HuR/CMTM6/PD-L1 axis.

Circular RNAs (circRNAs) have been shown to play diverse biological functions in the progression of multiple diseases. However, the impacts of circRNAs on breast cancer (BC) progression remains unclear. Therefore, the objective of this paper is to investigate the role and mechanisms of a functional circRNA in BC metastasis and immune escape. This study used a circRNA microarray and identified a novel circRNA hsa_circ_0067842. The validation and characteristics of hsa_circ_0067842 were investigated using qRT-PCR, sanger sequencing, RNase R treatment, actinomycin D treatment and fluorescence in situ hybridization (FISH). Gain- and loss-of-function assays were performed to evaluate the biological function of hsa_circ_0067842 in BC progression and immune escape. Mechanistically, the interaction between hsa_circ_0067842 and HuR was explored by RNA pull down, mass spectrometry (MS), subcellular component protein extraction and immunofluorescence (IF). The regulatory mechanisms of hsa_circ_0067842/HuR/CMTM6/PD-L1 axis were investigated by qRT-PCR, western blot, FISH, immunoprecipitation and rescue assays. The expression of hsa_circ_0067842 was upregulated in BC tissues and cells, which was found to be significantly associated with poor prognosis, regardless of other clinical covariates. Function assays showed that hsa_circ_0067842 promoted the migration and invasion capacities of BC cells. Moreover, co-culture experiment with peripheral blood mononuclear cells (PBMCs) showed that hsa_circ_0067842 played a role in the immune escape of BC cells. Mechanistically, our study showed that hsa_circ_0067842 interacted with HuR, affecting its nuclear translocation, thus enhancing the stability of CMTM6. CMTM6 not only enhances the migration and invasion ability of BC cells, but also affects the ubiquitination of PD-L1 and inhibits its degradation. Collectively, our results demonstrated that hsa_circ_0067842 promoted BC progression through the HuR/CMTM6/PD-L1 axis, providing new insight and a potential target for BC prognosis and therapy.

LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

The focus of the study is to examine the function of TYMSOS in immune escape of breast cancer, which is the most frequently diagnosed malignancy among women globally. Our study demonstrated that upregulated TYMSOS was associated with unfavorable prognosis and immune escape in breast cancer. TYMSOS promoted the malignant phenotypes of breast cancer cells, and reduced the cytotoxicity of NK92 cells on these cells. CBX3 was a downstream effector in TYMSOS-induced malignant phenotypes in breast cancer cells. Mechanistic studies showed that TYMSOS facilitated CBX3-mediated transcriptional repression of ULBP3, and it also promoted SYVN1-mediated ubiquitin-proteasomal degradation of ULBP3. TYMSOS promoted cell growth, metastasis, and immune escape via CBX3/ULBP3 or SYVN1/ULBP3 axis. The invivo studies further showed that silencing of TYMSOS repressed tumor growth and boosted NK cell cytotoxicity. In sum, TYMSOS boosted breast cancer metastasis and immune escape via CBX3/ULBP3 or SYVN1/ULBP3 axis.

HNRNPC promotes collagen fiber alignment and immune evasion in breast cancer via activation of the VIRMA-mediated TFAP2A/DDR1 axis

BackgroundCancers aggressively reorganize collagen in their microenvironment, leading to the evasion of tumor cells from immune surveillance. However, the biological significance and molecular mechanism of collagen alignment in breast cancer (BC) have not been well established.MethodsIn this study, BC-related RNA-Seq data were obtained from the TCGA database to analyze the correlation between DDR1 and immune cells. Mouse BC cells EO771 were selected for in vitro validation, and dual-luciferase experiments were conducted to examine the effect of TFAP2A on DDR1 promoter transcription activity. ChIP experiments were performed to assess TFAP2A enrichment on the DDR1 promoter, while Me-RIP experiments were conducted to detect TFAP2A mRNA m6A modification levels, and PAR-CLIP experiments were conducted to determine VIRMA’s binding to TFAP2A mRNA and RIP experiments to investigate HNRNPC’s recognition of m6A modification on TFAP2A mRNA. Additionally, an in vivo mouse BC transplant model and the micro-physiological system was constructed for validation, and Masson staining was used to assess collagen fiber arrangement. Immunohistochemistry was conducted to identify the number of CD8-positive cells in mouse BC tumors and Collagen IV content in ECM, while CD8 + T cell migration experiments were performed to measure CD8 + T cell migration.ResultsBioinformatics analysis showed that DDR1 was highly expressed in BC and negatively correlated with the proportion of anti-tumor immune cell infiltration. In vitro cell experiments indicated that VIRMA, HNRNPC, TFAP2A, and DDR1 were highly expressed in BC cells. In addition, HNRNPC promoted TFAP2A expression and, therefore, DDR1 transcription by recognizing the m6A modification of TFAP2A mRNA by VIRMA. In vivo animal experiments further confirmed that VIRMA and HNRNPC enhanced the TFAP2A/DDR1 axis, promoting collagen fiber alignment, reducing anti-tumor immune cell infiltration, and promoting immune escape in BC.ConclusionThis study demonstrated that HNRNPC promoted DDR1 transcription by recognizing VIRMA-unveiled m6A modification of TFAP2A mRNA, which enhanced collagen fiber alignment and ultimately resulted in the reduction of anti-tumor immune cell infiltration and promotion of immune escape in BC.

PARP9 knockdown confers protection against chemoresistance and immune escape of breast cancer cells by blocking the PI3K/AKT pathway.

This study probes the mechanism of the PARP9/PI3K/AKT/PD-L1 axis in the chemoresistance and immune escape of breast cancer cells. The expression of related genes was detected in MCF-7/FUL cells. After MCF-7/FUL cells were treated with sh-PARP9 and/or the PI3K/AKT pathway activator, drug resistance, proliferation, migration, invasion, and apoptosis were measured. Afterward, MCF-7/FUL cells were co-cultured with CD8+ T cells to examine the positive rate and density of MCF-7/FUL cells, the percentage and apoptosis of CD8+ T cells, and the expression of immune-related factors in cell supernatants. Nude mice were subcutaneously injected with sh-PARP9-transfected MCF-7/FUL cells for in vivo validation. PARP9 was highly expressed in MCF-7/FUL cells. Sh-PARP9 transfection suppressed cell migration, proliferation, and invasion while accelerating apoptosis in MCF-7/FUL cells, accompanied by downregulated PD-L1, p-PI3K, and p-AKT expression, and reduced IC50 and FUL resistance. After co-culture of MCF-7/FUL cells with CD8+ T cells, the percentage of CD8+ T cells, the expression of immune-related factors in supernatants, and the positive rate of MCF-7/FUL cells increased, while the apoptosis of CD8+ T cells and the density of adherent MCF-7/FUL cells were diminished. These trends were negated by further activating the PI3K/AKT pathway. PARP9 knockdown suppressed xenograft growth, decreased p-PI3K, p-AKT, PD-L1, and cyclin D1 expression, and augmented p-Cdc2 and cleaved caspase 3 levels in nude mice. PARP9 knockdown blocked the PI3K/AKT pathway to downregulate PD-L1, thus depressing chemoresistance and immune escape in breast cancer.

Metabolomic and Mitochondrial Fingerprinting of the Epithelial-to-Mesenchymal Transition (EMT) in Non-Tumorigenic and Tumorigenic Human Breast Cells.

Epithelial-to-mesenchymal transition (EMT) is key to tumor aggressiveness, therapy resistance, and immune escape in breast cancer. Because metabolic traits might be involved along the EMT continuum, we investigated whether human breast epithelial cells engineered to stably acquire a mesenchymal phenotype in non-tumorigenic and H-RasV12-driven tumorigenic backgrounds possess unique metabolic fingerprints. We profiled mitochondrial-cytosolic bioenergetic and one-carbon (1C) metabolites by metabolomic analysis, and then questioned the utilization of different mitochondrial substrates by EMT mitochondria and their sensitivity to mitochondria-centered inhibitors. "Upper" and "lower" glycolysis were the preferred glucose fluxes activated by EMT in non-tumorigenic and tumorigenic backgrounds, respectively. EMT in non-tumorigenic and tumorigenic backgrounds could be distinguished by the differential contribution of the homocysteine-methionine 1C cycle to the transsulfuration pathway. Both non-tumorigenic and tumorigenic EMT-activated cells showed elevated mitochondrial utilization of glycolysis end-products such as lactic acid, β-oxidation substrates including palmitoyl-carnitine, and tricarboxylic acid pathway substrates such as succinic acid. Notably, mitochondria in tumorigenic EMT cells distinctively exhibited a significant alteration in the electron flow intensity from succinate to mitochondrial complex III as they were highly refractory to the inhibitory effects of antimycin A and myxothiazol. Our results show that the bioenergetic/1C metabolic signature, the utilization rates of preferred mitochondrial substrates, and sensitivity to mitochondrial drugs significantly differs upon execution of EMT in non-tumorigenic and tumorigenic backgrounds, which could help to resolve the relationship between EMT, malignancy, and therapeutic resistance in breast cancer.

Combined Dusp4 and p53 loss with Dbf4 amplification drives tumorigenesis via cell cycle restriction and replication stress escape in breast cancer

AimDeregulated signaling pathways are a hallmark feature of oncogenesis and driver of tumor progression. Dual specificity protein phosphatase 4 (DUSP4) is a critical negative regulator of the mitogen-activated protein kinase (MAPK) pathway and is often deleted or epigenetically silenced in tumors. DUSP4 alterations lead to hyperactivation of MAPK signaling in many cancers, including breast cancer, which often harbor mutations in cell cycle checkpoint genes, particularly in TP53.MethodsUsing a genetically engineered mouse model, we generated mammary-specific Dusp4-deleted primary epithelial cells to investigate the necessary conditions in which DUSP4 loss may drive breast cancer oncogenesis.ResultsWe found that Dusp4 loss alone is insufficient in mediating tumorigenesis, but alternatively converges with loss in Trp53 and MYC amplification to induce tumorigenesis primarily through chromosome 5 amplification, which specifically upregulates Dbf4, a cell cycle gene that promotes cellular replication by mediating cell cycle checkpoint escape.ConclusionsThis study identifies a novel mechanism for breast tumorigenesis implicating Dusp4 loss and p53 mutations in cellular acquisition of Dbf4 upregulation as a driver of cellular replication and cell cycle checkpoint escape.

Tumor promoting effect of circ_002172 associates with induced immune escape in breast cancer via the miR-296-5p/CXCL12 axis

Comprehending the biology of tumorigenesis needs identification of determinants of the immune reaction during cancer development. This study intends to illustrate the mechanistic actions of a bioinformatically predicted circRNA circ_002172 in cytotoxic T lymphocytes (CTL) infiltration and escape of breast cancer (BC) from immunological destruction. Expression patterns of circ_002172, miR-296-5p, and CXCL12 were determined in BC tissues and cells. Effects of circ_002172, miR-296-5p, and CXCL12 on cell viability, migration, and invasion were examined through artificial modulation of their expression. The role of circ_002172 and CXCL12 on tumorigenesis was validated in subcutaneously transplanted and orthotopically transplanted tumors in nude mice. Upregulation of circ_002172 and CXCL12 and downregulation of miR-296-5p occurred in BC tissues and cells. Circ_002172 promoted the oncogenic phenotypes of BC cells in vitro and growth of tumors in vivo, which was reversed by knockdown of CXCL12 expression. Circ_002172, as a miR-296-5p sponge, upregulated expression CXCL12. Moreover, Ectopic expression of circ_002172 inhibited cytotoxic T lymphocytes (CTL) infiltration to promote the immune escape of BC. In conclusion, the tumor-promoting role of circ_002172 in BC was achieved by inducing immune escape via the miR-296-5p/CXCL12 axis.

Nanomedicine as a Promising Tool to Overcome Immune Escape in Breast Cancer.

Breast cancer is the most common type of malignancy and leading cause of cancer death among women worldwide. Despite the current revolutionary advances in the field of cancer immunotherapy, clinical response in breast cancer is frequently below expectations, in part due to various mechanisms of cancer immune escape that produce tumor variants that are resistant to treatment. Thus, a further understanding of the molecular events underlying immune evasion in breast cancer may guarantee a significant improvement in the clinical success of immunotherapy. Furthermore, nanomedicine provides a promising opportunity to enhance the efficacy of cancer immunotherapy by improving the delivery, retention and release of immunostimulatory agents in targeted cells and tumor tissues. Hence, it can be used to overcome tumor immune escape and increase tumor rejection in numerous malignancies, including breast cancer. In this review, we summarize the current status and emerging trends in nanomedicine-based strategies targeting cancer immune evasion and modulating the immunosuppressive tumor microenvironment, including the inhibition of immunosuppressive cells in the tumor area, the activation of dendritic cells and the stimulation of the specific antitumor T-cell response.

Deregulated molecules and pathways in the predisposition and dissemination of breast cancer cells to bone

BackgroundBone metastasis is the most common metastatic destination in advanced breast cancer, presenting a poor prognosis and clinical challenges in management. To date, the mechanism of bone metastasis in breast cancer remains largely unclear. MethodsDifferentially expressed genes in primary tumours that developed bone metastases were systematically analysed using both TCGA-BRCA and E-MTAB-4003 databases. Adaptive phenotype in the subsequent bone lesions was analysed in the GSE46161 database. A series of biomarkers including homing, immune escape, angiogenesis, and factors involved in both osteoblastogenesis and osteoclastogenesis were included to dissect the molecular events underlying bone metastasis in breast cancer. ResultsUpregulated expressions of GDF11 expression is positively correlated with colonization, osteoblastogenesis and osteoclastogenesis, whilst CD151 is positively associated with angiogenesis and immune escape. PAFAH1B2 expression is inversely correlated with the angiogenic process. Reduced YTHDF2 may facilitate cancer cell homing, osteoclastogenesis and immune escape in breast cancer. DPP9, FAS, ZNF519, RPP14 and FAU were evaluated for their potential involvement in for the homing to bone, escaping from immune surveillance, angiogenesis, osteoblastic activity and osteoclastic activity in the multi-step process of bone metastasis. ConclusionGDF11, CD151, PAFAH1B2 and YTHDF2 may play a pivotal role in the predisposition of metastasis to the bone from breast cancer, whilst DPP9, FAS, ZNF519, RPP14 and FAU may be actively involved in the adaptative colonisation of metastatic breast cancer cells in bone.

Progranulin induces immune escape in breast cancer via up-regulating PD-L1 expression on tumor-associated macrophages (TAMs) and promoting CD8+ T cell exclusion

BackgroundProgranulin (PGRN), as a multifunctional growth factor, is overexpressed in multiple tumors, but the role of PGRN on tumor immunity is still unclear. Here, we studied the effect of PGRN on breast cancer tumor immunity and its possible molecular mechanism.MethodsThe changes of macrophage phenotypes after PGRN treatment were detected by western blot, quantitative polymerase chain reaction (PCR) and flow cytometry. Western blot was used to study the signal molecular mechanism of PGRN regulating this process. The number and localization of immune cells in Wild-type (WT) and PGRN−/− breast cancer tissues were analyzed by immunohistochemical staining and immunofluorescence techniques. The activation and proliferation of CD8+ T cells were measured by flow cytometry.ResultsAfter being treated with PGRN, the expressions of M2 markers and programmed death ligand 1 (PD-L1) on macrophages increased significantly. Signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitor Stattic significantly inhibited the expression of PD-L1 and M2 related markers induced by PGRN. In WT group, CD8 were co-localized with macrophages and PD-L1, but not tumor cells. The number of immune cells in PGRN−/− breast cancer tissue increased, and their infiltration into tumor parenchyma was also enhanced. Moreover, in the co-culture system, WT peritoneal macrophages not only reduced the ratio of activated CD8+ T cells but also reduced the proportion of proliferating CD8+ T cells. The addition of programmed death receptor 1 (PD-1) and PD-L1 neutralizing antibodies effectively reversed this effect and restored the immune function of CD8+ T cells.ConclusionThese results demonstrate that PGRN promotes M2 polarization and PD-L1 expression by activating the STAT3 signaling pathway. Furthermore, through PD-1/PD-L1 interaction, PGRN can promote the breast tumor immune escape. Our research may provide new ideas and targets for clinical breast cancer immunotherapy.

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4+, CD8+ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3+ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.

Abstract A15: Immune-related changes in breast cancer tumor evolution

Abstract Immunotherapy is a highly promising therapeutic option in metastatic disease albeit only in a subset of patients possibly due to heterogeneity in the mechanisms by which tumors escape immune surveillance. Immune cells shape tumor evolution directly (e.g., anti-tumor immune response) and indirectly (e.g., changing the microenvironment) by selecting for cancer cells with certain properties. We hypothesized that the in situ (DCIS) to invasive ductal carcinoma (IDC) transition is a critical tumor progression step for immune escape in breast cancer that defines subsequent tumor evolution. In DCIS, cancer cells are physically separated from the stroma by the basement membrane and myoepithelial cell layer, and tumor-infiltrating leukocytes are rarely detected in direct contact with cancer cells. In contrast, in IDC, cancer cells and leukocytes are intermingled, thus, only cancer cells that can survive in this environment will play a role in disease progression. To dissect mechanisms of immune escape in breast cancer, we first analyzed the composition of leukocytes in normal breast tissues, DCIS, and IDC by polychromatic FACS. We found that DCIS and IDC contained significantly higher numbers of leukocytes, compared to normal breast, whereas in normal tissues more leukocytes were in the stromal than in the epithelial fraction. We also observed significant differences in the relative frequencies of several CD45+ cell types including increased neutrophils and decreased CD8+/CD4+ T cell ratios in tumors compared to normal stroma. Next, we analyzed the gene expression profiles of CD45+CD3+ T cells and found gene set enrichment of cytotoxic cells in DCIS including CD8+ T cells and NKT cells when compared to IDC. Conversely, we found enrichment for gene sets corresponding to regulatory T cells in IDC when compared to DCIS. Overall this suggested that DCIS had a more activated immune environment and IDC a more suppressed immune environment. We further explored this result by immunofluorescence (IF) and found fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including a set of matched DCIS and locally recurrent IDC tissues. We also found that the TCR clonotype was more diverse in DCIS than in normal breast and IDCs. Interestingly we detected a few relatively frequent clones that were shared among different DCIS, one of which was previously shown to recognize a protein from the Epstein-Bar virus. To elucidate mechanisms of immune evasion in IDC, we performed IF analysis of immune checkpoint proteins PD-L1 and TIGIT and found significant differences between DCIS and IDC. TIGIT-expressing T cells were more slightly frequent in DCIS than in IDC. PD-L1 expression was higher in the epithelial cancer cells in triple negative IDC compared to DCIS, and amplification of CD274 (encoding PD-L1) was only detected in triple negative IDCs. Given the close proximity of ERBB2 (encoding HER2) to a cluster of genes encoding several chemokines, we analyzed the HER2+ samples from the TCGA. We found that co-amplification of 17q12 chemokine cluster (CC) with ERBB2 was enriched in HER+ER+ luminal-like tumors but not in the HER2+ER breast tumors. We also found higher expression of both T cell activation and inhibition-related genes in tumors that lack CC gain. Also by assessing tumor samples by multicolor FISH and IF, we determined that there is an inverse correlation between CC amplification and activation of CD8+ T cells. Overall our results show co-evolution of cancer cells and the immune microenvironment during tumor progression. Citation Format: Carlos R. Gil del Alcazar, SungJin Huh, Muhammad B. Ekram, Anne Trinh, Lin L. Liu, Francisco Beca, Zi Xiaoyuan, Misuk Kwak, Helga Bergholtz, Ying Su, Lina Ding, Lina Ding, Hege G. Russnes, Andrea L. Richardson, Kirsten Babski, Elizabeth Min Hui Kim, Charles H. McDonnell, III, Jon Wagner, Ron Rowberry, Gordon Freeman, Deborah Dillon, Therese Sorlie, Lisa M. Coussens, Judy E. Garber, Rong Fan, Kristie Bobolis, Joon Jeong, So Yeon Park, Franziska Michor, Kornelia Polyak. Immune-related changes in breast cancer tumor evolution [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A15.

Abstract IA14: Breast tumor evolution: The role of the microenvironment

Abstract The immune system and the tissue microenvironment shape tumor evolution by providing selection for cancer cells with certain phenotypes. Immunotherapy is one of the most promising current cancer therapies and one of the very few that has the potential to cure even advanced metastatic disease. However, in breast cancer its success thus far has been limited to a subset of triple-negative tumors. This could be due to the testing of a limited set of immune-regulatory agents, but also a consequence of heterogeneity among tumors for mechanisms by which they escape immune surveillance. We hypothesized that the in situ to invasive carcinoma transition is a critical step for immune escape in breast cancer, since in DCIS (ductal carcinoma in situ) cancer cells are still physically separated from the stroma by the basement membrane and myoepithelial cell layer, and tumor-infiltrating leukocytes are rarely detected in direct contact with cancer cells. In contrast, in invasive tumors cancer cells and leukocytes are mixed, providing a strong selection for cancer cells that can overcome a potential immune attack. To begin dissecting mechanisms of immune escape during breast tumor evolution, we analyzed the composition of leukocytes in normal breast tissues, DCIS, and invasive ductal carcinomas (IDC) by a combination of technologies including FACS, RNA-seq, multicolor immunofluorescence, and immune-FISH. Based on these analyses, we found that invasive tumors have a less active immune environment due to a combination of upregulation of immune-suppressive mechanisms by both the cancer cells and various other cell types composing the microenvironment. Our results will improve our understanding of immune escape in breast cancer and aid the design of more effective immunotherapies for both advanced and early-stage disease. Citation Format: Kornelia Polyak. Breast tumor evolution: The role of the microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA14.

Abstract B62: Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in DCIS (ductal carcinoma in situ)

Abstract The normal mammary gland is composed of multiple cell types, including luminal and myoepithelial cells. Myoepithelial cells have also been referred to as natural tumor suppressors due to their inhibitory effect on various neoplastic phenotypes, including tumor cell growth, invasion, and angiogenesis. We have previously determined that DCIS-associated myoepithelial cells lack clonal genetic aberrations, but their DNA methylation and gene expression patterns are distinct from that of normal myoepithelial cells, implying perturbed differentiation. We also showed that myoepithelial cells play key roles in preventing tumor growth and invasive progression in experimental models of DCIS, and in immune escape in breast cancer. These data support the hypothesis that the progressive loss of normal myoepithelial cell function in DCIS is a key step in the transition to invasive breast carcinomas, and assessing myoepithelial markers may identify patients with high risk of progression. However, the mechanisms underlying myoepithelial cell aberrations and eventual loss in DCIS are not well understood. In the present study, we used a combination of genomic profiling of human tissue samples and functional assays in a DCIS model to investigate regulators of normal myoepithelial cells and perturbations of these in BRCA mutation carriers and in DCIS. We first explored cellular heterogeneity of the CD10+ cell population in normal human breast tissues of nulliparous and parous women, from reduction mammoplasties and BRCA1 and BRCA2 mutation carriers undergoing prophylactic mastectomy. We identified two distinct CD10+ cell populations distinguished by the expression of CD44 and characterized their gene expression profiles. We also profiled CD10+ cells from BRCA1 and BRCA2 mutation carriers compared to normal noncarriers and found a clear separation of three distinct groups reflecting germline mutation status. Next we defined the p63 cistrome and the enhancer landscape of normal myoepithelial cells and identified alterations of these due to germline BRCA mutation status. Based on our initial results, we focused in p63 and TCF7 as these two transcription factors appear to be central for establishing myoepithelial cell fate. To address the functional relevance of p63 and TCF7 in myoepithelial cell differentiation, we used the MCFDCIS xenograft model of DCIS. These results support the hypothesis that myoepithelial cells play key roles in invasive progression and have significant impact on our understanding of DCIS progression and the increased breast cancer risk of BRCA mutation carriers. Citation Format: Lina Ding, Ying Su, Muhammad Ekram, Sung Jin Huh, Noga Bloushtain-Qimron, Sibgat Choudhury, William Hines, Jun Yao, Mina Bissell, Kornelia Polyak. Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in DCIS (ductal carcinoma in situ) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B62.

Juxtacrine Signaling Inhibits Antitumor Immunity by Upregulating PD-L1 Expression.

Programmed death-ligand 1 (PD-L1) is a well-known immune checkpoint protein that helps cancer cells evade immune response. Anti-PD-L1 immune therapy has been approved for the treatment of several advanced human cancers. Therefore, further understanding of the regulatory mechanisms of PD-L1 is critical to improve PD-L1-targeting immunotherapy. Recent studies indicated that contact-dependent pathways may regulate anticancer immunity, highlighting the importance of cell contact-induced signaling in cancer immunity. Here, we show that tumor cell contact upregulates PD-L1 expression and reduces T-cell-mediated cell killing through the membrane receptor tyrosine kinase ephrin receptor A10 (EphA10), which is not expressed in normal tissues except testis and is known to mediate cell contact-dependent juxtacrine signaling. Knockout of EphA10 in tumor cells increased T-cell-mediated antitumor immunity in syngeneic mouse models. EphA10 expression also correlated positively with PD-L1 in human breast tumor tissues. Together, our data reveal that in addition to paracrine/autocrine signaling, cell contact-mediated juxtacrine signaling also promotes PD-L1 expression, implying that tumor cells may escape immune surveillance via this mechanism and that targeting EphA10 to boost antitumor immunity may be a new immune checkpoint blockade strategy for female patients with breast cancer.Significance: Regulation of PD-L1 expression by cell contact-mediated signaling promotes immune escape in breast cancer and may lead to the development of an immunotherapy with less adverse effects in female patients. Cancer Res; 78(14); 3761-8. ©2018 AACR.

Immune Escape in Breast Cancer During In Situ to Invasive Carcinoma Transition.

To investigate immune escape during breast tumor progression, we analyzed the composition of leukocytes in normal breast tissues, ductal carcinoma in situ (DCIS), and invasive ductal carcinomas (IDC). We found significant tissue and tumor subtype-specific differences in multiple cell types including T cells and neutrophils. Gene expression profiling of CD45+CD3+ T cells demonstrated a decrease in CD8+ signatures in IDCs. Immunofluorescence analysis showed fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including in matched DCIS and recurrent IDC. T-cell receptor clonotype diversity was significantly higher in DCIS than in IDCs. Immune checkpoint protein TIGIT-expressing T cells were more frequent in DCIS, whereas high PD-L1 expression and amplification of CD274 (encoding PD-L1) was only detected in triple-negative IDCs. Coamplification of a 17q12 chemokine cluster with ERBB2 subdivided HER2+ breast tumors into immunologically and clinically distinct subtypes. Our results show coevolution of cancer cells and the immune microenvironment during tumor progression.Significance: The design of effective cancer immunotherapies requires the understanding of mechanisms underlying immune escape during tumor progression. Here we demonstrate a switch to a less active tumor immune environment during the in situ to invasive breast carcinoma transition, and identify immune regulators and genomic alterations that shape tumor evolution. Cancer Discov; 7(10); 1098-115. ©2017 AACR.See related commentary by Speiser and Verdeil, p. 1062This article is highlighted in the In This Issue feature, p. 1047.