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BACKGROUND: The cut-off of < 1% positive cells to define estrogen receptor (ER) negativity by immunohistochemistry (IHC) in breast cancer (BC) is debated. We explored the tumor immune microenvironment and gene-expression profile of patients with early-stage HER2-negative ER-low (ER 1-9%) BC, comparing them to ER-negative (ER < 1%) and ER-intermediate (ER 10-50%) tumors. PATIENTS AND METHODS: Among 921 patients with early-stage I-III, ER ≤ 50%, HER2-negative BCs, tumors were classified as ER-negative (n = 712), ER-low (n = 128), or ER-intermediate (n = 81). Tumor-infiltrating lymphocytes (TILs) were evaluated. CD8+, FOXP3+ cells, and PD-L1 status were assessed by IHC and quantified by digital pathology. We analyzed 776 BC-related genes in 116 samples. All tests were 2-sided at < 0.05 significance level. RESULTS: ER-low and ER-negative tumors exhibited similar median TILs, significantly higher than ER-intermediate tumors. CD8/FOXP3 ratio and PD-L1 positivity rates were comparable between ER-low and ER-negative groups. These groups showed similar enrichment in Basal-like intrinsic subtypes and comparable expression of immune-related genes. ER-low and ER-intermediate tumors showed significant transcriptomic differences. High TILs (≥30%) were associated with improved relapse-free survival (RFS) in ER-low (5-year RFS 78.6% vs 66.2%, log-rank p = .033, hazard ratio (HR) 0.37 [95% CI 0.15-0.96]) and ER-negative patients (5-year RFS 85.2% vs 69.8%, log-rank p < .001, HR 0.41 [95% CI 0.27-0.60]). CONCLUSIONS: ER-low and ER-negative tumors are similar biological and molecular entities, supporting their comparable clinical outcomes and treatment responses, including to immunotherapy. Our findings contribute to the growing evidence calling for a reevaluation of ER-positive BC classification and management, aligning ER-low and ER-negative tumors more closely.
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The transcription factor receptor-interacting protein 140 (RIP140) regulates intestinal homeostasis and tumorigenesis through Wnt signaling. In this study, we investigated its effect on the Notch/HES1 signaling pathway. In colorectal cancer (CRC) cell lines, RIP140 positively regulated HES1 gene expression at the transcriptional level via a recombining binding protein suppressor of hairless (RBPJ)/neurogenic locus notch homolog protein 1 (NICD)-mediated mechanism. In support of these in vitro data, RIP140 and HES1 expression significantly correlated in mouse intestine and in a cohort of CRC samples, thus supporting the positive regulation of HES1 gene expression by RIP140. Interestingly, when the Notch pathway is fully activated, RIP140 exerted a strong inhibition of HES1 gene transcription controlled by the level of HES1 itself. Moreover, RIP140 directly interacts with HES1 and reversed its mitogenic activity in human CRC cells. In line with this observation, HES1 levels were associated with a better patient survival only when tumors expressed high levels of RIP140. Our data identify RIP140 as a key regulator of the Notch/HES1 signaling pathway, with a dual effect on HES1 gene expression at the transcriptional level and a strong impact on colon cancer cell proliferation.
Subject(s)
Cell Proliferation , Colonic Neoplasms , Gene Expression Regulation, Neoplastic , Nuclear Receptor Interacting Protein 1 , Transcription Factor HES-1 , Animals , Humans , Mice , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Nuclear Receptor Interacting Protein 1/metabolism , Receptors, Notch/metabolism , Receptors, Notch/genetics , Signal Transduction , Transcription Factor HES-1/metabolism , Transcription Factor HES-1/geneticsABSTRACT
INTRODUCTION: Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. METHODS: Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. RESULTS: Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. CONCLUSIONS: F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.
Subject(s)
Antineoplastic Agents , Benzamides , Nitriles , Phenylthiohydantoin , Triple Negative Breast Neoplasms , Humans , Animals , Mice , Triple Negative Breast Neoplasms/pathology , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Cathepsin D , Mice, Nude , Cell Line, Tumor , Antibody-Dependent Cell Cytotoxicity , Antineoplastic Agents/therapeutic use , Killer Cells, Natural , Immunoglobulin Fc FragmentsABSTRACT
There exists a variety of studies about tumor-infiltrating immune cells (TIICs) in cervical cancer, but their prognostic value in correlation with the histopathological subtype has never been investigated. Therefore, the aim of this study was to quantify TIICs in a panel of 238 sporadic cervical cancers and investigate the correlation with cervical cancer subtype and patient survival. TIICs levels were significantly increased in the subgroup of CSCC (191 samples) in comparison to CAC (47 samples). In CSCC, TIICs' infiltration showed a negative correlation with age, FIGO stage and with the histone protein modification H3K4me3. Moreover, in CAC, it was positively correlated with p16 and with the glucocorticoid receptor and inversely correlated with the MDM2 protein and with H3K4me3. Interestingly, immune infiltration was an independent positive prognosticator for disease-free survival (DFS) in patients with CSCC, those bearing tumors with the strongest TIICs infiltration showing the better DFS. Altogether, the present study provides a differentiated overview of the relations between TIIC levels and prognosis in patients with CSCC vs. patients with CAC.
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BACKGROUND: Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. METHODS: Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The "one-two punch" sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. RESULTS: We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3ß/Wnt/ß-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. CONCLUSION: Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a "one-two punch" approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.
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Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutic agents. The current mechanistic evidence from cell-based studies suggests that the matricellular protein SPARC has a tumor-promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited. Here, we analyzed by immunohistochemistry the prognostic value of tumor and stromal cell SPARC expression in 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). We also quantified PD-L1 and PD-1 expression. We detected SPARC expression in tumor cells (42.4%), cancer-associated fibroblasts (CAFs; 88.1%), tumor-associated macrophages (77.1%), endothelial cells (75.2%) and tumor-infiltrating lymphocytes (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. Multivariate analysis showed that SPARC expression in CAFs was an independent prognostic factor. We also detected tumor and stromal cell SPARC expression in TNBC cytosols, and in patient-derived xenografts and cell lines. Furthermore, we analyzed publicly available single-cell mRNA sequencing data and found that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved in tumor-related processes. We then showed that fibroblast-secreted SPARC had a tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our study demonstrates that SPARC expression in CAFs is an independent prognostic marker of poor outcome in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC targeted therapy.
Subject(s)
Antineoplastic Agents , Cancer-Associated Fibroblasts , Triple Negative Breast Neoplasms , Humans , Prognosis , Triple Negative Breast Neoplasms/pathology , Cancer-Associated Fibroblasts/metabolism , Endothelial Cells/metabolism , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Osteonectin/genetics , Osteonectin/metabolismABSTRACT
Background: T cell immunoreceptor with Ig and ITIM domains (TIGIT) interacts with poliovirus receptor (PVR) to contribute to cancer immune escape. Recently, TIGIT and PVR have been identified as promising immunotherapy targets. Their gene expression is upregulated in many solid tumors, but their protein expression level is not well documented, particularly in triple negative breast cancer (TNBC), the breast cancer subtype that most benefit from immunotherapy. Methods: TIGIT and PVR expression levels were assessed by immunohistochemistry in 243 surgically resected localized TNBC and then their relationship with clinical-pathological features and clinical outcome was analyzed. Results: TIGIT expression was observed in immune cells from the tumor microenvironment, whereas PVR was mainly expressed by tumor cells. High TIGIT expression was significantly associated with age (p=0.010), histological grade (p=0.014), non-lobular histology (p=0.024), adjuvant chemotherapy (p=0.006), and various immune cell populations (tumor infiltrating lymphocytes (TILs), CD3+, CD8+, PD-1+ cells; all p<0.0001), PD-L1+ tumor cells (p<0.0001), and PD-L1+ stromal cells (p=0.003). Infiltration by TIGIT+ cells tended to be higher in non-molecular apocrine tumors (p=0.088). PVR was significantly associated with histological grade (p<0.0001), the basal-like (p=0.003) and non-molecular apocrine phenotypes (p=0.039), high TILs infiltration (p=0.011), CD3+ (p=0.002), CD8+ (p=0.024) T cells, and PD-L1 expression in tumor (p=0.003) and stromal cells (p=0.001). In univariate analysis, only known prognostic factors (age, tumor size, lymph node status, adjuvant chemotherapy, TILs and CD3+ T-cell infiltrate) were significantly associated with relapse-free survival (RFS) and overall survival. High TIGIT and PVR expression levels tended to be associated with longer RFS (p=0.079 and 0.045, respectively). The analysis that included only non-molecular apocrine TNBC revealed longer RFS for tumors that strongly expressed TIGIT or PVR (p=0.025 for TIGIT and 0.032 for PVR). Conclusions: These results indicated that in TNBC, TIGIT+ cells can easily interact with PVR to exert their inhibitory effects. Their wide expression in TNBC and their association with other immune checkpoint components suggest the therapeutic interest of the TIGIT-PVR axis.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/pathology , Prognosis , B7-H1 Antigen/metabolism , Neoplasm Recurrence, Local , Receptors, Immunologic/genetics , Tumor MicroenvironmentABSTRACT
The prognostic impact of tumor-infiltrating lymphocytes (TILs) is intensively investigated in breast cancer (BC). It is already known that triple-negative breast cancer (TNBC), the most aggressive type of BC, has the highest percentage of TILs. In addition, there is an influence of steroid hormone receptor expression (type I nuclear receptors) on TIL subpopulations in breast cancer tissue. The link between type II nuclear receptors and the level of TILs is unclear. Therefore, the aim of this study was to quantify TILs in a panel of 264 sporadic breast cancers and investigate the correlation of TIL levels with type I and II nuclear receptors expression. TIL levels were significantly increased in the subgroup of TNBC. By contrast, they decreased in estrogen (ER)- or progesterone receptor (PR)-positive cases. Moreover, TIL levels were correlated with type II nuclear receptors, including PPARγ, with a significant inverse correlation of the nuclear form (r = −0.727, p < 0.001) and a weak positive correlation of the cytoplasmic form (r = 0.202, p < 0.002). Surprisingly, BC cases with a TIL Salgado score of >15% showed a significantly decreased overall survival. In addition, peritumoral inflammation was also quantified in BC tissue samples. In our cohort, although the level of peritumoral inflammation was not correlated with OS, it determined the prognostic value of ER, PR, and PPARγ in BC. Altogether, the present study provides a differentiated overview of the relations between nuclear receptor expression, TIL levels, peritumoral inflammation, and prognosis in BC.
ABSTRACT
Background: Triple-negative breast cancers (TNBCs) have a worse prognosis, but might respond to immunotherapies. Macrophages are plastic cells that can adopt various phenotypes and functions. Although they are a major immune population in TNBCs, the relationship between tumor-associated macrophages (TAMs) and TNBC progression has been rarely explored, with controversial results. Methods: We evaluated the prognostic impact of TAMs, quantified by immunohistochemistry with anti-CD68, -IRF8, -CD163, and -CD206 antibodies, in a well-described cohort of 285 patients with non-metastatic TNBC. Results: CD68 (p = 0.008), IRF8 (p = 0.001), and CD163 (p < 0.001) expression positively correlated with higher tumor grade, while CD206 was associated with smaller tumor size (p < 0.001). All macrophage markers were associated with higher tumor-infiltrating lymphocyte numbers and PD-L1 expression. Univariate survival analyses reported a significant positive correlation between CD163+ or CD206+ TAMs and relapse-free survival (respectively: HR = 0.52 [0.28−0.97], p = 0.027, and HR = 0.51 [0.31−0.82], p = 0.005), and between CD206+ TAMs and overall survival (HR = 0.54 [0.35−0.83], p = 0.005). In multivariate analysis, there was a trend for an association between CD206+ TAMs and relapse-free survival (HR = 0.63 [0.33−1.04], p = 0.073). Conclusions: These data suggest that CD206 expression defines a TAM subpopulation potentially associated with favorable outcomes in patients with TNBC. CD206 expression might identify an immune TNBC subgroup with specific therapeutic options.
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Urokinase plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI1), have been reported as prognostic and predictive biomarkers in breast cancer, particularly in patients with nodenegative tumors. uPA and PAI1 expression levels classify patients into a poorprognosis subgroup, requiring adjuvant chemotherapy and a favorableprognosis subgroup, which can be considered for deescalation. However, the clinical use of these two biomarkers remains limited, since freshfrozen/fresh tumor samples are currently required for their quantification. The aim of the present study was to compare PLAU and SERPINE1 mRNA expression levels (corresponding to uPA and PAI1 proteins, respectively), assessed using in situ hybridization in 83 formalinfixed paraffinembedded (FFPE) breast tumor samples, with uPA and PAI1 protein expression assessed using immunometric assay with paired freshfrozen breast cancer samples. The results from the two methods significantly correlated as regards uPA quantification; however, >30% of the samples were discordant, according to the clinically validated threshold. Concordance between the two analytical methods was less prominent for PAI1 protein and SERPINE1 mRNA. Taken together, the results of the present study indicate that although PLAU and SERPINE1 mRNA may be reliably detected in FFPE samples using in situ hybridization, this technology cannot be used as a substitute for the replacement of the immunometric assayderived quantification on freshfrozen samples.
Subject(s)
Breast Neoplasms , Urokinase-Type Plasminogen Activator , Breast Neoplasms/pathology , Female , Formaldehyde , Humans , In Situ Hybridization , Membrane Proteins , Paraffin Embedding , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
HER2-low breast cancer (i.e., HER 1+ or 2+, without gene amplification) is an emerging subtype for which very few data are available, especially within the triple-negative breast cancer (TNBC) group. Our aim was to evaluate HER2 expression and its prognostic value in a large retrospective series of patients with non-metastatic TNBC (median age: 57.7 years; range: 28.5-98.6). Among the 296 TNBC samples, 83.8% were HER2 0, 13.5% were HER2 1+, and 2.7% were HER2 2+ (HercepTestTM and 2018 ASCO/CAP guidelines for HER2 scoring). CK5/6 and/or EGFR-expressing androgen receptors and FOXA1-expressing tumors were classified as basal-like (63.8%) and molecular apocrine-like (MA, 40.2%), respectively. Compared with HER2 0 tumors, HER2 1+/2+ tumors exhibited a lower histological grade (1/2) (35.4% vs. 18.2%, p = 0.007) and MA profile (57.5% vs. 36.7%, p = 0.008). Moreover, patients with HER2 1+/2+ tumors were older (p = 0.047). After a median follow-up of 9.7 years, HER2 2+ tumors (compared with HER2 0/1+ tumors) were associated with worse relapse-free survival (RFS) (HR = 3.16, 95% CI [1.27; 7.85], p = 0.034) in a univariate analysis. Overall survival (OS) and RFS were not different in the HER2 0 and 1+/2+ groups. HER2 levels were not significantly associated with OS or RFS in a multivariate analysis.
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Microsatellite instability (MSI) is related to the alteration of mismatch repair (MMR) genes and plays a key role in colorectal cancer (CRC) pathogenesis. We previously reported that the transcription factor Nuclear Receptor Interacting Protein 1 (NRIP1) is involved in sporadic intestinal tumorigenesis. The aim of this study was to decipher its role in MSI CRC. By using different mouse models and engineered cell lines, we demonstrated that NRIP1 increased MSH2 and MSH6 MMR gene transcription and mRNA/protein levels. In human CRC cells, NRIP1 expression was associated with decreased MSI and the hypermutator phenotype, and with resistance to chemotherapy drugs. Using a cohort of 194 CRC patients, we detected in 22% of the cases a MSI-induced frameshift mutation in the NRIP1 coding sequence. This genetic alteration generates a truncated protein with a dominant negative activity that increased human CRC cell proliferation and impaired the regulation of MSH2 and MSH6 gene expression. Moreover, the NRIP1 mutant correlated with a decreased overall survival of patients with advanced CRC, especially when MLH1-deficient. By decreasing the expression of MSH2 and MSH6 gene expression, the NRIP1 variant may amplify MLH1-dependent CRC progression and behave as a new prognostic marker of advanced MSI CRC.
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Chemokines and their receptors are key players in breast cancer progression and outcome. Previous studies have shown that the chemokine receptor CXCR2 was expressed at higher levels by cells of the tumor microenvironment in triple-negative breast cancers (TNBCs). The aim of this study was to focus our attention on a retrospective cohort of 290 TNBC cases and analyze the involvement of CXCR2, CD11b (a marker of granulocytes) and CD66b (a marker of neutrophils) and their link with immune infiltration and immune checkpoint markers. We report that high densities of CXCR2-, CD11b- and CD66b-positive cells were associated with high-grade tumors. Moreover, molecular apocrine TNBCs, defined here as tumors that express both AR and FOXA1 biomarkers, exhibited low levels of CXCR2 and CD11b. High CXCR2 and CD11b levels were correlated with elevated density of tumor-infiltrating lymphocytes (TILs), CD8+ cytotoxic lymphocytes, expression of PD-L1 by tumor and stromal cells and of PD-1 by stromal cells. On the other hand, CD66b levels were associated only with CD8+, stromal PD-L1 and PD-1 expression. In univariate analysis, low levels of CXCR2 were correlated with poor OS and RFS. In multivariate analysis, low levels of CXCR2 were associated with poor OS. Finally, in TNBC treated with adjuvant chemotherapy, CXCR2 density was associated with longer RFS. Overall, our data highlight the potential beneficial association of high levels of CXCR2 with a subgroup of TNBC patients characterized by a better prognosis.
ABSTRACT
Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/- knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.
Subject(s)
Cathepsin D/metabolism , Extracellular Matrix/metabolism , Neoplasm Proteins/metabolism , Osteonectin/metabolism , Peptide Fragments/pharmacology , Triple Negative Breast Neoplasms/pathology , Tumor Microenvironment , Amino Acid Sequence , Animals , Binding Sites , Cathepsin D/deficiency , Cathepsin D/genetics , Cell Adhesion , Female , Fibroblasts , Gene Expression Regulation, Neoplastic , Humans , Hydrogen-Ion Concentration , Mammary Neoplasms, Experimental/enzymology , Mice , Mice, Knockout , Mice, Transgenic , Molecular Weight , Neoplasm Invasiveness , Neoplasm Proteins/genetics , Osteonectin/genetics , Peptide Fragments/metabolism , Protein Domains , Proteolysis , Substrate Specificity , Transendothelial and Transepithelial Migration , Triple Negative Breast Neoplasms/enzymologyABSTRACT
Alterations to cell polarization or to intercellular junctions are often associated with epithelial cancer progression, including breast cancers (BCa). We show here that the loss of the junctional scaffold protein MAGI1 is associated with bad prognosis in luminal BCa, and promotes tumorigenesis. E-cadherin and the actin binding scaffold AMOTL2 accumulate in MAGI1 deficient cells which are subjected to increased stiffness. These alterations are associated with low YAP activity, the terminal Hippo-pathway effector, but with an elevated ROCK and p38 Stress Activated Protein Kinase activities. Blocking ROCK prevented p38 activation, suggesting that MAGI1 limits p38 activity in part through releasing actin strength. Importantly, the increased tumorigenicity of MAGI1 deficient cells is rescued in the absence of AMOTL2 or after inhibition of p38, demonstrating that MAGI1 acts as a tumor-suppressor in luminal BCa by inhibiting an AMOTL2/p38 stress pathway.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Angiomotins/metabolism , Breast Neoplasms/prevention & control , Carcinogenesis/pathology , Cell Adhesion Molecules/metabolism , Guanylate Kinases/metabolism , Signal Transduction , Stress, Physiological , p38 Mitogen-Activated Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing/deficiency , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cadherins/metabolism , Carcinogenesis/metabolism , Cell Adhesion Molecules/deficiency , Cell Line, Tumor , Cell Proliferation , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Guanylate Kinases/deficiency , Humans , Phenotype , Protein Binding , YAP-Signaling Proteins/metabolism , beta Catenin/metabolism , rho-Associated Kinases/metabolismABSTRACT
The prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations in solid cancers is still debated. Here, we investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 patients with triple-negative breast cancer (TNBC). A high γδ T cell density (>6.625 γδ T cells/mm2) was associated with younger age (p = 0.008), higher tumor histological grade (p = 0.002), adjuvant chemotherapy (p = 0.010), BRCA1 promoter methylation (p = 0.010), TIL density (p < 0.001), and PD-L1 (p < 0.001) and PD-1 expression (p = 0.040). In multivariate analyses, γδ T cell infiltration (cutoff = 6.625 γδ T cells/mm2) was an independent prognostic factor (5-year relapse-free survival: 63.3% vs. 89.8%, p = 0.027; 5-year overall survival: 73.8% vs. 89.9%, p = 0.031, for low vs. high infiltration). This prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, but the difference was not significant in the subgroup with PIK3CA-mutated tumors. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a candidate prognostic tool in patients with TNBC.
ABSTRACT
In a previous phase II study (THERAPY), cetuximab and trastuzumab combination, as second-line after progression with gemcitabine, showed disease stabilization in 27% of 33 patients with pancreatic carcinoma. In the present phase II multicenter study, we assessed the efficacy and tolerance of gemcitabine, trastuzumab plus erlotinib as first-line treatment of metastatic pancreatic cancer. The primary endpoint was disease control rate (DCR, RECIST v.1); secondary endpoints were progression-free (PFS), overall (OS) survival and toxicity (NCI-CTCAE v3.0). Ancillary study addressed the predictive value of both EGFR/HER2 expression and KRAS mutational status. Sixty-three patients from four centers were included (62 evaluable for toxicity, 59 for efficacy), median age was 62 years (35-77), 59.7% men. The median treatment duration was 16.1 weeks (2.1-61). Eleven patients (19%) reported a partial tumor response, and 33 (56%) disease stabilization. DCR was 74.6% (95%CI: 61.8-85.0; 44/59 patients). After a median follow-up of 23.3 months (0.6-23.6), median PFS was 3.5 months (95%CI: 2.4-3.8) and median OS 7.9 months (95%CI: 5.1-10.2). PFS was significantly longer in patients with grade ≥ 2 cutaneous toxicities vs patients with grade 0-1 toxicities (HR = 0.55, 95%CI: 0.33-0.92, P = .020). Expression of EGFR and HER2 was correlated with PFS and OS in multivariate analysis; HER2 expression was correlated with the tumor response. Main severe toxicities were neutropenia (32%), cutaneous rash (37%) and thrombosis/embolisms (35.5%). This triplet combination is effective in terms of disease control, PFS and OS, and acceptable for safety. A larger study to investigate this combination compared to the standard regimen should be discussed.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Deoxycytidine/analogs & derivatives , Erlotinib Hydrochloride/administration & dosage , Pancreatic Neoplasms/drug therapy , Trastuzumab/administration & dosage , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Deoxycytidine/administration & dosage , Deoxycytidine/adverse effects , ErbB Receptors/genetics , Erlotinib Hydrochloride/adverse effects , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Neoplasm Metastasis , Pancreatic Neoplasms/genetics , Receptor, ErbB-2/genetics , Survival Analysis , Trastuzumab/adverse effects , Treatment Outcome , GemcitabineABSTRACT
To clarify the function of cyclin A2 in colon homeostasis and colorectal cancer (CRC), we generated mice deficient for cyclin A2 in colonic epithelial cells (CECs). Colons of these mice displayed architectural changes in the mucosa and signs of inflammation, as well as increased proliferation of CECs associated with the appearance of low- and high-grade dysplasias. The main initial events triggering those alterations in cyclin A2-deficient CECs appeared to be abnormal mitoses and DNA damage. Cyclin A2 deletion in CECs promoted the development of dysplasia and adenocarcinomas in a murine colitis-associated cancer model. We next explored the status of cyclin A2 expression in clinical CRC samples at the mRNA and protein levels and found higher expression in tumors of patients with stage 1 or 2 CRC compared with those of patients with stage 3 or 4 CRC. A meta-analysis of 11 transcriptome data sets comprising 2239 primary CRC tumors revealed different expression levels of CCNA2 (the mRNA coding for cyclin A2) among the CRC tumor subtypes, with the highest expression detected in consensus molecular subtype 1 (CMS1) and the lowest in CMS4 tumors. Moreover, we found high expression of CCNA2 to be a new, independent prognosis factor for CRC tumors.
Subject(s)
Colon/metabolism , Colorectal Neoplasms/metabolism , Cyclin A2/metabolism , Homeostasis , Neoplasm Proteins/metabolism , Neoplasms, Experimental/metabolism , Animals , Colon/pathology , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Cyclin A2/genetics , Mice , Mice, Knockout , Neoplasm Proteins/genetics , Neoplasm Staging , Neoplasms, Experimental/diagnosis , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , PrognosisABSTRACT
BACKGROUND: No treatment option was available for patients with RAS-mutated (RASmt) metastatic colorectal cancer (mCRC) who progress after standard combined chemotherapies at the time of the study. After promising results in phase II, the aim of the present NEXIRI-2/PRODIGE 27 trial was to assess the 2-month non-progression rate for sorafenib (NEX) plus irinotecan (IRI), that is, NEXIRI, treatment. METHODS: Patients with RASmt mCRC after failure of oxaliplatin, IRI, fluoropyrimidines, and bevacizumab were randomized between NEXIRI (IRI 120-180 mg/m2 intravenous, D1 = D15 plus oral NEX 400 mg twice a day) versus IRI (180 mg/m2) versus NEX. Primary endpoint was the 2-month non-progression rate. Secondary endpoints included progression-free and overall survival (PFS and OS), safety, and germline cyclin D1 (CCND1) rs9344 polymorphisms analyses. RESULTS: A total of 173 patients were included, 59 in NEXIRI, 57 in IRI, and 57 in NEX arms. The 2-month non-progression rate was 52.6% (95% confidence interval [CI]: 39%-66%), 21.4% (10%-33%), and 19.3% (9%-30%) for NEXIRI, IRI, and NEX. Median PFS was 3.6 (95% CI: 2-4.2), 1.7 (1.7-1.8), and 2 (1.8-2.3) months and the median OS was 7.2 (5.8-9.4), 6.3 (4.8-8), and 5.6 (3.9-7.7) months for NEXIRI, IRI, and NEX, respectively. For NEXIRI rs9344CCND1 A/A genotype patients, OS was 19.6 months (95% CI: 4.8-not reached). Main grade 3 toxicities included neutropenia, febrile neutropenia, diarrhea, hand-foot syndrome, and hypertension. CONCLUSIONS: In patients with RASmt mCRC who progressed after standard combined chemotherapies, the results of 2-month non-progression rate and median PFS in the NEXIRI arm were in favor of an increase of the time before progression.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Colorectal Neoplasms/drug therapy , Irinotecan/pharmacology , Sorafenib/pharmacology , ras Proteins/genetics , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Drug Resistance, Neoplasm/genetics , Female , Humans , Irinotecan/therapeutic use , Male , Middle Aged , Mutation , Progression-Free Survival , Sorafenib/therapeutic use , Time FactorsABSTRACT
The tumor microenvironment appears essential in cancer progression and chemokines are mediators of the communication between cancer cells and stromal cells. We have previously shown that the ligands of the chemokine receptor CXCR2 were expressed at higher levels in triple-negative breast cancers (TNBC). Our hypothesis was that CXCR2 expression could also be altered in breast cancer. Here, we have analyzed the potential role of CXCR2 in breast cancer in a retrospective cohort of 105 breast cancer patients. Expression of CXCR2, CD11b (a marker of granulocytes) and CD66b (a marker of neutrophils) was analyzed by immunohistochemistry on tumor samples. We demonstrated that CXCR2 stained mainly stromal cells and in particular neutrophils. CXCR2, CD11b and CD66b expression were correlated with high grade breast cancers. Moreover, TNBC displayed a higher expression of CXCR2, CD11b and CD66b than hormone receptor positive or Her2 positive tumors. High levels of CXCR2 and CD11b, but not CD66b, were associated with a higher infiltration of T lymphocytes and B lymphocytes. We also observed a correlation between CXCR2 and AP-1 activity. In univariate analyses, CXCR2, but not CD11b or CD66b, was associated with a lower risk of relapse; CXCR2 remained significant in multivariate analysis. Our data suggest that CXCR2 is a stromal marker of TNBC. However, higher levels of CXCR2 predicted a lower risk of relapse.