RESUMEN
In view of the relatively limited efficacy of immunotherapies targeting the PD-1-PD-L1 axis in triple-negative breast cancer (TNBC) and of published reports on tumor-promoting roles of TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs), we determined the incidence of TNFR2+ TILs in TNBC patient tumors, their association with disease outcome and relations with PD-1+ TILs. Using a cohort of treatment-naïve TNBC patients with long follow-up (n = 70), we determined the presence of TNFR2+ TILs and PD-1+ TILs by immunohistochemistry. TILs (≥ 1% of cellular mass) and TNFR2+ TILs (≥ 1% of total TILs) were detected in 96% and 74% of tumors, respectively. The presence of TILs at > 5% of tumor cell mass ("Positive TILs"), as well as of positive TNFR2+ TILs (> 5%), was independently associated with good prognosis, and combination of both parameters demonstrated superior outcome relative to their lower levels. PD1+ TILs (> 5/hot spot) were detected in 63% of patients. High levels of PD-1+ TILs (> 20/hot spot) showed an unfavorable disease outcome, and in their presence, the favorable outcome of positive TNFR2+ TILs was ablated. Thus, TNFR2+ TILs are strongly connected to improved prognosis in TNBC; these findings suggest that TNFR2+ TILs have favorable effects in TNBC patients, unlike the tumor-promoting roles attributed to them in other cancer systems. Overall, our observations propose that the TNFR2+ TIL subset should not be targeted in the course of TNBC therapy; rather, its beneficial impacts may become into power when anti-PD-1 regimens-that may potentiate immune activities-are administered to TNBC patients.
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Biomarcadores de Tumor/análisis , Carcinoma Ductal de Mama/mortalidad , Linfocitos Infiltrantes de Tumor/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Neoplasias de la Mama Triple Negativas/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Ductal de Mama/inmunología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Tasa de Supervivencia , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.
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Enzimas Desubicuitinizantes/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Selección Genética , Tioléster Hidrolasas/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Farmacorresistencia Bacteriana/genética , Escherichia coli/enzimología , Escherichia coli/metabolismo , Modelos Moleculares , Plásmidos , Transducción de Señal/genética , Ubiquitina/metabolismoRESUMEN
PD-L1 has been characterized as an inhibitory immune checkpoint, leading to the suppression of potential anti-tumor immune activities in many cancer types. In view of the relatively limited efficacy of immune checkpoint blockades against PD-L1 in breast cancer, our recent study addressed the possibility that in addition to its immune-inhibitory functions, PD-L1 promotes the pro-metastatic potential of the cancer cells themselves. Indeed, our published findings demonstrated that PD-L1 promoted pro-metastatic functions of breast cancer cells in a cell-autonomous manner, both in vitro and in vivo. These functions fully depended on the integrity of the S283 intracellular residue of PD-L1. Here, using siRNAs and the S283A-PD-L1 variant, we demonstrate that the cell-autonomous pro-metastatic functions of PD-L1-tumor cell proliferation and invasion, and release of the pro-metastatic chemokine CXCL8-required the activation of STAT3 and STAT1 in luminal A and triple-negative breast cancer cells. The cell-autonomous pro-metastatic functions of PD-L1 were potently impaired upon inhibition of N-linked glycosylation (kifunensine). Site-specific mutants at each of the N-linked glycosylation sites of PD-L1 (N35, N192, N200, and N219) revealed that they were all required for PD-L1-induced pro-metastatic functions to occur; the N219 site was the main regulator of STAT3 and STAT1 activation, with accompanying roles for N192 and N200 (depending on the cell type). Using a T cell-independent mouse system, we found that cells expressing N35A-PD-L1 and N219A-PD-L1 had a significantly lower tumorigenic and metastatic potential than cells expressing WT-PD-L1. TCGA analyses revealed significant associations between reduced survival and high levels of α-mannosidase II (inferring on N-linked glycosylation) in breast cancer patients. These findings suggest that N-linked glycosylation of PD-L1 may be used to screen for patients who are at greater risk of disease progression, and that modalities targeting N-linked glycosylated PD-L1 may lead to the inhibition of its cell-autonomous pro-metastatic functions and to lower tumor progression in breast cancer.
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Antígeno B7-H1 , Neoplasias de la Mama Triple Negativas , Ratones , Animales , Humanos , Glicosilación , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Neoplasias de la Mama Triple Negativas/patología , Linfocitos T , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Pro-inflammatory cytokines play key roles in elevating cancer progression in triple-negative breast cancer (TNBC). We demonstrate that specific combinations between TNFα, IL-1ß and IFNγ up-regulated the proportion of human TNBC cells co-expressing the inhibitory immune checkpoints PD-L1 and PD-L2: TNFα + IL-1ß in MDA-MB-231 cells and IFNγ + IL-1ß in BT-549 cells; in the latter cells, the process depended entirely on STAT1 activation, with no involvement of p65 (CRISPR-Cas9 experiments). Highly significant associations between the pro-inflammatory cytokines and PD-L1/PD-L2 expression were revealed in the TCGA dataset of basal-like breast cancer patients. In parallel, we found that the pro-inflammatory cytokines regulated the expression of the soluble receptors of tumor necrosis factor α (TNFα), namely sTNFR1 and sTNFR2; moreover, we revealed that sTNFR1 and sTNFR2 serve as anti-metastatic and protective factors in TNBC, reducing the TNFα-induced production of inflammatory pro-metastatic chemokines (CXCL8, CXCL1, CCL5) by TNBC cells. Importantly, we found that in the context of inflammatory stimulation and also without exposure to pro-inflammatory cytokines, elevated levels of PD-L1 have down-regulated the production of anti-tumor sTNFR1 and sTNFR2. These findings suggest that in addition to its immune-suppressive activities, PD-L1 may promote disease course in TNBC by inhibiting the protective effects of sTNFR1 and sTNFR2.
RESUMEN
Therapies targeting the PD-L1/PD-1 axis have recently been introduced to triple-negative breast cancer (TNBC) with limited efficacy, suggesting that this axis promotes tumor progression through mechanisms other than immune suppression. Here, we over-expressed WT-PD-L1 in human TNBC cells (express endogenous PD-L1) and in luminal-A breast cancer cells (no endogenous PD-L1 expression) and demonstrated that cell-autonomous PD-L1 activities lead to increased tumor cell growth, invasion and release of pro-metastatic factors (CXCL8, sICAM-1, GM-CSF). These activities were promoted by PD-1 and were inhibited by mutating S283 in PD-L1. Invasion of WT-PD-L1-cells required signaling by chemokine receptors CXCR1/2, CCR2 and CCR5 through autocrine circuits involving CXCL8, CCL2 and CCL5. Studies with T cell-deficient mice demonstrated that cell-autonomous WT-PD-L1 activities in TNBC cells increased tumor growth and metastasis compared to knock-out (KO)-PD-L1-cells, whereas S283A-PD-L1-expressing cells had minimal ability to form tumors and did not metastasize. Overall, our findings reveal autonomous and PD-1-induced tumor-promoting activities of PD-L1 that depend on S283 and on chemokine circuits. These results suggest that TNBC patients whose tumors express PD-L1 could benefit from therapies that prevent immune suppression by targeting PD-1/CTLA-4, alongside with antibodies to PD-L1, which would allow maximal impact by mainly targeting the cancer cells.
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The authors apologize for several typos/technical inaccuracies in the original article [...].
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Triple-negative breast cancer (TNBC) is primarily treated via chemotherapy; in parallel, efforts are made to introduce immunotherapies into TNBC treatment. CD4+ TNFR2+ lymphocytes were reported as Tregs that contribute to tumor progression. However, our published study indicated that TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs) were associated with improved survival in TNBC patient tumors. Based on our analyses of the contents of CD4+ and CD8+ TILs in TNBC patient tumors, in the current study, we determined the impact of chemotherapy on CD4+ and CD8+ TIL subsets in TNBC mouse tumors. We found that chemotherapy led to (1) a reduction in CD4+ TNFR2+ FOXP3+ TILs, indicating that chemotherapy decreased the content of CD4+ TNFR2+ Tregs, and (2) an elevation in CD8+ TNFR2+ and CD8+ TNFR2+ PD-1+ TILs; high levels of these two subsets were significantly associated with reduced tumor growth. In spleens of tumor-bearing mice, chemotherapy down-regulated CD4+ TNFR2+ FOXP3+ cells but the subset of CD8+ TNFR2+ PD-1+ was not present prior to chemotherapy and was not increased by the treatment. Thus, our data suggest that chemotherapy promotes the proportion of protective CD8+ TNFR2+ TILs and that, unlike other cancer types, therapeutic strategies directed against TNFR2 may be detrimental in TNBC.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Estudios de Cohortes , Supervivencia sin Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , PronósticoRESUMEN
Cellular heterogeneity poses an immense therapeutic challenge in cancer due to a constant change in tumor cell characteristics, endowing cancer cells with the ability to dynamically shift between states. Intra-tumor heterogeneity is largely driven by cancer cell plasticity, demonstrated by the ability of malignant cells to acquire stemness and epithelial-to-mesenchymal transition (EMT) properties, to develop therapy resistance and to escape dormancy. These different aspects of cancer cell remodeling are driven by intrinsic as well as by extrinsic signals, the latter being dominated by factors of the tumor microenvironment. As part of the tumor milieu, chronic inflammation is generally regarded as a most influential player that supports tumor development and progression. In this review article, we put together recent findings on the roles of inflammatory elements in driving forward key processes of tumor cell plasticity. Using breast cancer as a representative research system, we demonstrate the critical roles played by inflammation-associated myeloid cells (mainly macrophages), pro-inflammatory cytokines [such as tumor necrosis factor α (TNFα) and interleukin 6 (IL-6)] and inflammatory chemokines [primarily CXCL8 (interleukin 8, IL-8) and CXCL1 (GROα)] in promoting tumor cell remodeling. These inflammatory components form a common thread that is involved in regulation of the three plasticity levels: stemness/EMT, therapy resistance, and dormancy. In view of the fact that inflammatory elements are a common denominator shared by different aspects of tumor cell plasticity, it is possible that their targeting may have a critical clinical benefit for cancer patients.