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BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples. METHODS: We performed genome-wide epigenetic mapping of DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmc) using oxidative bisulfite sequencing from formalin-embedded sections. We identified overlap with transcriptional signatures in formalin-fixed, paraffin-embedded tissue from resected patients, via bioinformatics using iCluster and mutational profiling and confirmed them in vivo. RESULTS: We found that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci, including GATA6, which promote differentiated classical pancreatic subtypes. We showed that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5-methylcytosine hydroxylase TET2. Furthermore, we found that SMAD4 directly supports TET2 levels in classical pancreatic tumors, and loss of SMAD4 expression was associated with reduced 5hmc, GATA6, and squamous-like tumors. Importantly, enhancing TET2 stability using metformin and vitamin C/ascorbic acid restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. CONCLUSIONS: We identified epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data showed that restoring epigenetic control increases biomarkers of classical pancreatic tumors that are associated with improved therapeutic responses and survival.
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5-Metilcitosina/análogos & derivados , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Dioxigenasas/metabolismo , Epigénesis Genética , Factor de Transcripción GATA6/genética , Neoplasias Pancreáticas/genética , Transcripción Genética , 5-Metilcitosina/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácido Ascórbico/farmacología , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/enzimología , Carcinoma Ductal Pancreático/patología , Diferenciación Celular , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Epigénesis Genética/efectos de los fármacos , Epigenoma , Epigenómica , Factor de Transcripción GATA6/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Metformina/farmacología , Ratones Desnudos , Ratones Transgénicos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Estudios Retrospectivos , Proteína Smad4/genética , Proteína Smad4/metabolismo , Transcripción Genética/efectos de los fármacos , Transcriptoma , Vía de Señalización Wnt/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This Letter describes the continued SAR exploration of small molecule Legumain inhibitors with the aim of developing a potent and selective in vitro tool compound. Work continued in this Letter explores the use of alternative P2-P3 linker units and the P3 group SAR which led to the identification of 10t, a potent, selective and cellularly active Legumain inhibitor. We also demonstrate that 10t has activity in both cancer cell viability and colony formation assays.
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Cisteína Endopeptidasas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Supervivencia Celular/efectos de los fármacos , Cisteína Endopeptidasas/química , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Estructura MolecularRESUMEN
Here, we show for the first time, that the familial breast/ovarian cancer susceptibility gene BRCA1 activates the Notch pathway in breast cells by transcriptional upregulation of Notch ligands and receptors in both normal and cancer cells. We demonstrate through chromatin immunoprecipitation assays that BRCA1 is localized to a conserved intronic enhancer region within the Notch ligand Jagged-1 (JAG1) gene, an event requiring ΔNp63. We propose that this BRCA1/ΔNp63-mediated induction of JAG1 may be important the regulation of breast stem/precursor cells, as knockdown of all three proteins resulted in increased tumoursphere growth and increased activity of stem cell markers such as Aldehyde Dehydrogenase 1 (ALDH1). Knockdown of Notch1 and JAG1 phenocopied BRCA1 knockdown resulting in the loss of Estrogen Receptor-α (ER-α) expression and other luminal markers. A Notch mimetic peptide could activate an ER-α promoter reporter in a BRCA1-dependent manner, whereas Notch inhibition using a γ-secretase inhibitor reversed this process. We demonstrate that inhibition of Notch signalling resulted in decreased sensitivity to the anti-estrogen drug Tamoxifen but increased expression of markers associated with basal-like breast cancer. Together, these findings suggest that BRCA1 transcriptional upregulation of Notch signalling is a key event in the normal differentiation process in breast tissue.
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Proteína BRCA1/fisiología , Neoplasias de la Mama/genética , Mama/metabolismo , Receptores Notch/genética , Animales , Mama/citología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Proteínas de Unión al Calcio/genética , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Antagonistas de Estrógenos/farmacología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Células MCF-7 , Proteínas de la Membrana/genética , Ratones , Receptor Notch1/genética , Receptores Notch/biosíntesis , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/genética , Tamoxifeno/farmacología , Factores de Transcripción/fisiología , Transcripción Genética , Proteínas Supresoras de Tumor/fisiología , Regulación hacia ArribaRESUMEN
This Letter describes the further development and SAR exploration of a novel series of Legumain inhibitors. Based upon a previously identified Legumain inhibitor from our group, we explored the SAR of the carbamate phenyl ring system to probe the P3 pocket of the enzyme. This led to the identification of a sub-nanomolar inhibitor of Legumain.
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Compuestos de Bifenilo/farmacología , Carbamatos/farmacología , Cisteína Endopeptidasas/metabolismo , Inhibidores Enzimáticos/farmacología , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/química , Carbamatos/síntesis química , Carbamatos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is considered a non-immunogenic tumor, and immune checkpoint inhibitor monotherapy lacks efficacy in this disease. Radiotherapy (RT) can stimulate the immune system. Here, we show that treatment of KPC and Pan02 murine PDAC cells with RT and gemcitabine upregulated PD-L1 expression in a JAK/Stat1-dependent manner. In vitro, PD-L1 inhibition did not alter radio- and chemosensitivity. In vivo, addition of anti-PD-L1 to high (12, 5 × 3, 20 Gy) but not low (6, 5 × 2 Gy) RT doses significantly improved tumor response in KPC and Pan02 allografts. Radiosensitization after PD-L1 blockade was associated with reduced CD11b+Gr1+ myeloid cell infiltration and enhanced CD45+CD8+ T-cell infiltration with concomitant upregulation of T-cell activation markers including CD69, CD44, and FasL, and increased CD8:Treg ratio. Depletion of CD8+ T cells abrogated radiosensitization by anti-PD-L1. Blockade of PD-L1 further augmented the effect of high RT doses (12 Gy) in preventing development of liver metastases. Exploring multiple mathematical models reveals a mechanism able to explain the observed synergy between RT and anti-PD-L1 therapy. Our findings provide a rationale for testing the use of immune checkpoint inhibitors with RT in PDAC.
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Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/radioterapia , Antígeno B7-H1/antagonistas & inhibidores , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/radioterapia , Desoxicitidina/análogos & derivados , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Animales , Linfocitos T CD8-positivos , Desoxicitidina/administración & dosificación , Modelos Animales de Enfermedad , Ratones , Modelos Teóricos , Resultado del Tratamiento , GemcitabinaRESUMEN
Gemcitabine constitutes one of the backbones for chemotherapy treatment in pancreatic ductal adenocarcinoma (PDAC), but patients often respond poorly to this agent. Molecular markers downstream of gemcitabine treatment in preclinical models may provide an insight into resistance mechanisms. Using cytokine arrays, we identified potential secretory biomarkers of gemcitabine resistance (response) in the transgenic KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) mouse model of PDAC. We verified the oncogenic role of the cytokine tissue inhibitor of matrix metalloproteinases 1 (TIMP1) in primary pancreatic tumors and metastases using both in vitro techniques and animal models. We identified potential pathways affected downstream of TIMP1 using the Illumina Human H12 array. Our findings were validated in both primary and metastatic models of pancreatic cancer. Gemcitabine increased inflammatory cytokines including TIMP1 in the KPC mouse model. TIMP1 was upregulated in patients with pancreatic intraepithelial neoplasias grade 3 and PDAC lesions relative to matched normal pancreatic tissue. In addition, TIMP1 played a role in tumor clonogenic survival and vascular density, while TIMP1 inhibition resensitized tumors to gemcitabine and radiotherapy. We observed a linear relationship between TIMP-1 expression, liver metastatic burden, and infiltration by CD11b+Gr1+ myeloid cells and CD4+CD25+FOXP3+ Tregs, whereas the presence of tumor cells was required for immune cell infiltration. Overall, our results identify TIMP1 upregulation as a resistance mechanism to gemcitabine and provide a rationale for combining chemo/radiotherapy with TIMP1 inhibitors in PDAC. Cancer Res; 77(21); 5952-62. ©2017 AACR.
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Desoxicitidina/análogos & derivados , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidor Tisular de Metaloproteinasa-1/genética , Animales , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Desoxicitidina/farmacología , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/secundario , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/radioterapia , Interferencia de ARN , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Carga Tumoral/efectos de la radiación , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
INTRODUCTION: Preclinical CT-guided radiotherapy platforms are increasingly used but the CT images are characterized by poor soft tissue contrast. The aim of this study was to develop a robust and accurate method of MRI-guided radiotherapy (MR-IGRT) delivery to abdominal targets in the mouse. METHODS: A multimodality cradle was developed for providing subject immobilisation and its performance was evaluated. Whilst CT was still used for dose calculations, target identification was based on MRI. Each step of the radiotherapy planning procedure was validated initially in vitro using BANG gel dosimeters. Subsequently, MR-IGRT of normal adrenal glands with a size-matched collimated beam was performed. Additionally, the SK-N-SH neuroblastoma xenograft model and the transgenic KPC model of pancreatic ductal adenocarcinoma were used to demonstrate the applicability of our methods for the accurate delivery of radiation to CT-invisible abdominal tumours. RESULTS: The BANG gel phantoms demonstrated a targeting efficiency error of 0.56 ± 0.18 mm. The in vivo stability tests of body motion during MR-IGRT and the associated cradle transfer showed that the residual body movements are within this MR-IGRT targeting error. Accurate MR-IGRT of the normal adrenal glands with a size-matched collimated beam was confirmed by γH2AX staining. Regression in tumour volume was observed almost immediately post MR-IGRT in the neuroblastoma model, further demonstrating accuracy of x-ray delivery. Finally, MR-IGRT in the KPC model facilitated precise contouring and comparison of different treatment plans and radiotherapy dose distributions not only to the intra-abdominal tumour but also to the organs at risk. CONCLUSION: This is, to our knowledge, the first study to demonstrate preclinical MR-IGRT in intra-abdominal organs. The proposed MR-IGRT method presents a state-of-the-art solution to enabling robust, accurate and efficient targeting of extracranial organs in the mouse and can operate with a sufficiently high throughput to allow fractionated treatments to be given.
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Neoplasias Abdominales/diagnóstico por imagen , Neoplasias Abdominales/radioterapia , Imagen por Resonancia Magnética/métodos , Planificación de la Radioterapia Asistida por Computador/métodos , Radioterapia Guiada por Imagen/métodos , Abdomen/diagnóstico por imagen , Abdomen/efectos de la radiación , Glándulas Suprarrenales/diagnóstico por imagen , Glándulas Suprarrenales/efectos de la radiación , Animales , Línea Celular Tumoral , Humanos , Imagen por Resonancia Magnética/instrumentación , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Endogámicos NOD , Ratones Desnudos , Ratones Transgénicos , Movimiento (Física) , Imagen Multimodal/instrumentación , Trasplante de Neoplasias , Fantasmas de Imagen , Radiometría/instrumentación , Dosificación Radioterapéutica , Planificación de la Radioterapia Asistida por Computador/instrumentación , Radioterapia Guiada por Imagen/instrumentación , Tomografía Computarizada por Rayos X/instrumentación , Tomografía Computarizada por Rayos X/métodos , Carga TumoralRESUMEN
The unrestrained proliferation of cancer cells requires a high level of ribosome biogenesis. The first stage of ribosome biogenesis is the transcription of the large ribosomal RNAs (rRNAs); the structural and functional components of the ribosome. Transcription of rRNA is carried out by RNA polymerase I (Pol-I) and its associated holoenzyme complex.Here we report that BRCA1, a nuclear phosphoprotein, and a known tumour suppressor involved in variety of cellular processes such as DNA damage response, transcriptional regulation, cell cycle control and ubiquitylation, is associated with rDNA repeats, in particular with the regulatory regions of the rRNA gene.We demonstrate that BRCA1 interacts directly with the basal Pol-I transcription factors; upstream binding factor (UBF), selectivity factor-1 (SL1) as well as interacting with RNA Pol-I itself. We show that in response to DNA damage, BRCA1 occupancy at the rDNA repeat is decreased and the observed BRCA1 interactions with the Pol-I transcription machinery are weakened.We propose, therefore, that there is a rDNA associated fraction of BRCA1 involved in DNA damage dependent regulation of Pol-I transcription, regulating the stability and formation of the Pol-I holoenzyme during initiation and/or elongation in response to DNA damage.
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Proteína BRCA1/metabolismo , ARN Polimerasa I/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína BRCA1/genética , Línea Celular , Línea Celular Tumoral , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Humanos , Células MCF-7 , Unión Proteica , Interferencia de ARN , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic stroma. We examined the prognostic value of stroma density and activity in patients with resectable PDAC treated with surgery and adjuvant gemcitabine-based chemotherapy. FFPE-tissue from the pancreatectomy of 145 patients was immunohistochemically stained for haematoxylin-eosin and Masson's trichrome to assess stroma density, and alpha-smooth muscle actin (αSMA) expression for activated pancreatic stellate cells. Their expression was correlated with clinicopathological characteristics as well as overall survival (OS), progression-free survival (PFS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS). After a mean follow-up of 20 months (range, 2-69 months), the median OS was 21 months and the 3-year OS was 35.7%. In multivariate analysis, highly-dense stroma was an independent prognostic parameter for OS (p = 0.001), PFS (p = 0.007), LPFS (p = 0.001) and DMFS (p = 0.002), while αSMA expression lacked significance. Interestingly, highly-dense stroma retained significance for the four clinical endpoints only in early (pT1-2) but not late (pT3-4) stage tumors. Additionally, late pT-stage (pT3-4), the presence of lymph node metastases (pN+ vs pN0), perineural/neural invasion and administration of adjuvant chemotherapy also correlated with prognosis in multivariate analysis. Altogether, stroma density constitutes an independent prognostic marker in PDAC patients treated with adjuvant chemotherapy. Our findings highlight the dynamic complexity of desmoplasia and indicate that highly-dense stroma is correlated with better outcome. Further validation of the prognostic value of stroma as a biomarker and its role in PDAC patients after adjuvant chemotherapy is warranted and will be performed in a prospective study.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/secundario , Pancreatectomía/mortalidad , Neoplasias Pancreáticas/patología , Células del Estroma/patología , Anciano , Carcinoma Ductal Pancreático/terapia , Terapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Metástasis Linfática , Masculino , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias Pancreáticas/terapia , Pronóstico , Tasa de Supervivencia , Neoplasias PancreáticasRESUMEN
We examined the prognostic value of programmed cell death-1 (PD-1) and its ligand (PD-L1) together with CD8+ tumor-infiltrating lymphocytes (TILs) and FOXP3+ Tregs in resectable pancreatic ductal adenocarcinoma (PDAC) samples treated with adjuvant chemotherapy. Whole-mount FFPE tissue sections from 145 pancreatectomies were immunohistochemically stained for PD-1, PD-L1, CD8 and FOXP3. Their expression was correlated with clinicopathological characteristics, and overall survival (OS), progression-free survival (PFS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), in the context of stroma density (haematoxylin-eosin) and activity (alpha-smooth muscle actin) and in regard to intratumoral lymphoid aggregates. The median OS was 21 months after a mean follow-up of 20 months (range, 2-69 months). In multivariate analysis, high PD-1+ TILs expression was associated with better OS (p = 0.049), LPFS (p = 0.017) and DMFS (p = 0.021). Similar findings were observed for CD8+ TILs, whereas FOXP3 and PD-L1 lacked prognostic significance. Although TIL distribution was heterogeneous, tumors of high stroma density had higher infiltration of CD8+ TILs than loose density stroma and vice versa (p < 0.001), whereas no correlation was found with stromal activity. Sixty (41.4%) tumors contained lymphoid aggregates and the presence of PD-1+ TILs was associated with better OS (p = 0.030), LPFS (p = 0.025) and DMFS (p = 0.033), whereas CD8+ TILs only correlated with superior LPFS (p = 0.039). PD-1+ and CD8+ TILs constitute independent prognostic markers in patients with PDAC treated with adjuvant chemotherapy. Our study provides important insight on the role of PD-1/PD-L1 in the context of desmoplastic stroma and could help guide future immunotherapies in PDAC.
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Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/metabolismo , Antígenos CD8/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Factores de Transcripción Forkhead/metabolismo , Neoplasias Pancreáticas/diagnóstico , Receptor de Muerte Celular Programada 1/metabolismo , Células del Estroma/metabolismo , Adolescente , Adulto , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Valor Predictivo de las Pruebas , Pronóstico , Células del Estroma/patología , Análisis de Supervivencia , Adulto JovenRESUMEN
We assessed the prognostic value of hypoxia (carbonic anhydrase 9; CA9), vessel density (CD31), with macrophages (CD68) and B cells (CD20) that can interact and lead to immune suppression and disease progression using scanning and histological mapping of whole-mount FFPE pancreatectomy tissue sections from 141 primarily resectable pancreatic ductal adenocarcinoma (PDAC) samples treated with surgery and adjuvant chemotherapy. Their expression was correlated with clinicopathological characteristics, and overall survival (OS), progression-free survival (PFS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), also in the context of stroma density (haematoxylin-eosin) and activity (alpha-smooth muscle actin). The median OS was 21 months after a mean follow-up of 20 months (range, 2-69 months). The median tumor surface area positive for CA9 and CD31 was 7.8% and 8.1%, respectively. Although total expression of these markers lacked prognostic value in the entire cohort, nevertheless, high tumor compartment CD68 expression correlated with worse PFS (p = 0.033) and DMFS (p = 0.047). Also, high CD31 expression predicted for worse OS (p = 0.004), PFS (p = 0.008), LPFS (p = 0.014) and DMFS (p = 0.004) in patients with moderate density stroma. High stromal and peripheral compartment CD68 expression predicted for significantly worse outcome in patients with loose and moderate stroma density, respectively. Altogether, in contrast to the current notion, hypoxia levels in PDAC appear to be comparable to other malignancies. CD31 and CD68 constitute prognostic markers in patient subgroups that vary according to tumor compartment and stromal density. Our study provides important insight on the pathophysiology of PDAC and should be exploited for future treatments.
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Antígenos CD20/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Células del Estroma/metabolismo , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/terapia , Terapia Combinada , Femenino , Humanos , Hipoxia/metabolismo , Inmunohistoquímica , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neovascularización Patológica/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/terapia , Pronóstico , Análisis de Supervivencia , Neoplasias PancreáticasRESUMEN
TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.