Inactivation Of E-cadherin Research Articles

Insights into E-Cadherin Impairment in CDH1-Unaltered Invasive Lobular Carcinoma: A Comprehensive Bioinformatic Study.

Invasive lobular carcinoma exhibits unique morphological features frequently associated with alterations in CDH1. Although some studies have identified abnormalities in adhesion factors other than E-cadherin, the molecular mechanisms underlying E-cadherin abnormalities in CDH1-unaltered invasive lobular carcinoma remain poorly understood. In this study, we investigated the molecular underpinnings of E-cadherin dysregulation in invasive lobular carcinoma in the absence of CDH1 gene alterations, using comprehensive bioinformatic analyses. We conducted a comparative study of CDH1-mutated and non-mutated invasive lobular carcinoma and evaluated the differences in mRNA levels, reverse-phase protein array, methylation, and miRNAs. We observed that invasive lobular carcinoma cases without CDH1 alterations exhibited a significantly higher incidence of the Claudin-low subtype (p < 0.01). The results of the reverse-phase protein array indicate no significant difference in E-cadherin expression between CDH1-mutated and non-mutated cases. Therefore, abnormalities in E-cadherin production also exist in CDH1 non-mutated invasive lobular carcinoma. Considering that there are no differences in mRNA levels and methylation status, post-translational modifications are the most plausible explanation for the same. Hence, future studies should focus on elucidating the mechanism underlying E-cadherin inactivation via post-translational modifications in CDH1 non-mutated invasive lobular carcinoma.

CircEPB41L2 blocks the progression and metastasis in non-small cell lung cancer by promoting TRIP12-triggered PTBP1 ubiquitylation

The metastasis of non-small cell lung cancer (NSCLC) is the leading death cause of NSCLC patients, which requires new biomarkers for precise diagnosis and treatment. Circular RNAs (circRNAs), the novel noncoding RNA, participate in the progression of various cancers as microRNA or protein sponges. We revealed the mechanism by which circEPB41L2 (hsa_circ_0077837) blocks the aerobic glycolysis, progression and metastasis of NSCLC through modulating protein metabolism of PTBP1 by the E3 ubiquitin ligase TRIP12. With ribosomal RNA-depleted RNA seq, 57 upregulated and 327 downregulated circRNAs were identified in LUAD tissues. circEPB41L2 was selected due to its dramatically reduced levels in NSCLC tissues and NSCLC cells. Interestingly, circEPB41L2 blocked glucose uptake, lactate production, NSCLC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, acting as a scaffold, circEPB41L2 bound to the RRM1 domain of the PTBP1 and the E3 ubiquitin ligase TRIP12 to promote TRIP12-mediated PTBP1 polyubiquitylation and degradation, which could be reversed by the HECT domain mutation of TRIP12 and circEPB41L2 depletion. As a result, circEPB41L2-induced PTBP1 inhibition led to PTBP1-induced PKM2 and Vimentin activation but PKM1 and E-cadherin inactivation. These findings highlight the circEPB41L2-dependent mechanism that modulates the “Warburg Effect” and EMT to inhibit NSCLC development and metastasis, offering an inhibitory target for NSCLC treatment.

Canonical Kaiso target genes define a functional signature that associates with breast cancer survival and the invasive lobular carcinoma histological type.

Invasive lobular carcinoma (ILC) is a low- to intermediate-grade histological breast cancer type caused by mutational inactivation of E-cadherin function, resulting in the acquisition of anchorage independence (anoikis resistance). Most ILC cases express estrogen receptors, but options are limited in relapsed endocrine-refractory disease as ILC tends to be less responsive to standard chemotherapy. Moreover, ILC can relapse after >15 years, an event that currently cannot be predicted. E-cadherin inactivation leads to p120-catenin-dependent relief of the transcriptional repressor Kaiso (ZBTB33) and activation of canonical Kaiso target genes. Here, we examined whether an anchorage-independent and ILC-specific transcriptional program correlated with clinical parameters in breast cancer. Based on the presence of a canonical Kaiso-binding consensus sequence (cKBS) in the promoters of genes that are upregulated under anchorage-independent conditions, we defined an ILC-specific anoikis resistance transcriptome (ART). Converting the ART genes into human orthologs and adding published Kaiso target genes resulted in the Kaiso-specific ART (KART) 33-gene signature, used subsequently to study correlations with histological and clinical variables in primary breast cancer. Using publicly available data for ERPOS Her2NEG breast cancer, we found that expression of KART was positively associated with the histological ILC breast cancer type (p < 2.7E-07). KART expression associated with younger patients in all invasive breast cancers and smaller tumors in invasive ductal carcinoma of no special type (IDC-NST) (<2 cm, p < 6.3E-10). We observed associations with favorable long-term prognosis in both ILC (hazard ratio [HR] = 0.51, 95% CI = 0.29-0.91, p < 3.4E-02) and IDC-NST (HR = 0.79, 95% CI = 0.66-0.93, p < 1.2E-04). Our analysis thus defines a new mRNA expression signature for human breast cancer based on canonical Kaiso target genes that are upregulated in E-cadherin deficient ILC. The KART signature may enable a deeper understanding of ILC biology and etiology. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Modulation of E-Cadherin Function through the AmotL2 Isoforms Promotes Ameboid Cell Invasion.

The spread of tumor cells and the formation of distant metastasis remain the main causes of mortality in cancer patients. However, the mechanisms governing the release of cells from micro-environmental constraints remain unclear. E-cadherin negatively controls the invasion of epithelial cells by maintaining cell-cell contacts. Furthermore, the inactivation of E-cadherin triggers invasion in vitro. However, the role of E-cadherin is complex, as metastasizing cells maintain E-cadherin expression, which appears to have a positive role in the survival of tumor cells. In this report, we present a novel mechanism delineating how E-cadherin function is modulated to promote invasion. We have previously shown that E-cadherin is associated with p100AmotL2, which is required for radial actin formation and the transmission of mechanical force. Here, we present evidence that p60AmotL2, which is expressed in invading tumor cells, binds to the p100AmotL2 isoform and uncouples the mechanical constraint of radial actin filaments. We show for the first time that the coupling of E-cadherin to the actin cytoskeleton via p100AmotL2 is directly connected to the nuclear membrane. The expression of p60AmotL2 inactivates this connection and alters the properties of the nuclear lamina, potentiating the invasion of cells into micropores of the extracellular matrix. In summary, we propose that the balance of the two AmotL2 isoforms is important in the modulation of E-cadherin function and that an imbalance of this axis promotes ameboid cell invasion.

Results of a worldwide survey on the currently used histopathological diagnostic criteria for invasive lobular breast cancer

Invasive lobular carcinoma (ILC) represents the second most common subtype of breast cancer (BC), accounting for up to 15% of all invasive BC. Loss of cell adhesion due to functional inactivation of E-cadherin is the hallmark of ILC. Although the current world health organization (WHO) classification for diagnosing ILC requires the recognition of the dispersed or linear non-cohesive growth pattern, it is not mandatory to demonstrate E-cadherin loss by immunohistochemistry (IHC). Recent results of central pathology review of two large randomized clinical trials have demonstrated relative overdiagnosis of ILC, as only ~60% of the locally diagnosed ILCs were confirmed by central pathology. To understand the possible underlying reasons of this discrepancy, we undertook a worldwide survey on the current practice of diagnosing BC as ILC. A survey was drafted by a panel of pathologists and researchers from the European lobular breast cancer consortium (ELBCC) using the online tool SurveyMonkey®. Various parameters such as indications for IHC staining, IHC clones, and IHC staining procedures were questioned. Finally, systematic reporting of non-classical ILC variants were also interrogated. This survey was sent out to pathologists worldwide and circulated from December 14, 2020 until July, 1 2021. The results demonstrate that approximately half of the institutions use E-cadherin expression loss by IHC as an ancillary test to diagnose ILC and that there is a great variability in immunostaining protocols. This might cause different staining results and discordant interpretations. As ILC-specific therapeutic and diagnostic avenues are currently explored in the context of clinical trials, it is of importance to improve standardization of histopathologic diagnosis of ILC diagnosis.

LGR6-dependent conditional inactivation of E-cadherin and p53 leads to invasive skin and mammary carcinomas in mice

Tissue-specific inactivation of E-cadherin combined with tumor suppressor loss leads to invasive and metastatic cancers in mice. While epidermal E-cadherin loss in mice induces squamous cell carcinomas, inactivation of E-cadherin in the mammary gland leads to invasive lobular carcinoma. To further explore the carcinogenic consequences of cell-cell adhesion loss in these compartments, we developed a new conditional mouse model inactivating E-cadherin (Cdh1) and p53 (Trp53) simultaneously in cells expressing the leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6), a putative epithelial stem cell marker in the skin and alveolar progenitor marker in the mammary gland.Compound Lgr6-CreERT2;Cdh1F;Trp53F female mice containing either heterozygous or homozygous Cdh1F alleles were bred, and Lgr6-driven Cre expression was activated in pre-puberal mice using tamoxifen. We observed that 41% of the mice (16/39) developed mostly invasive squamous-type skin carcinomas, but also a non-lobular mammary tumor was formed. In contrast to previous K14cre or WAPcre E-cadherin and p53 compound models, no significant differences were detected in the tumor-free survival of Lgr6-CreERT2 heterozygous Cdh1F/WT;Trp53F/F versus homozygous Cdh1F/F;Trp53F/F mice (778 versus 754 days, p=0.5). One Cdh1F homozygous mouse presented with lung metastasis that originated from a non-lobular and ERα negative invasive mammary gland carcinoma with squamous metaplasia. In total, 2/8 (25%) Cdh1F heterozygous and 3/12 (25%) Cdh1F homozygous mice developed metastases to lungs, liver, lymph nodes, or the gastro-intestinal tract.In conclusion, we show that inducible and conditional Lgr6-driven inactivation of E-cadherin and p53 in mice causes squamous cell carcinomas of the skin in approximately 40% of the mice and an occasional ductal-type mammary carcinoma after long latency periods.

Targeted molecular profiling of salivary duct carcinoma with rhabdoid features highlights parallels to other apocrine and discohesive neoplasms: which phenotype should drive classification?

Salivary duct carcinoma with rhabdoid features (SDC-RF) is a recently-described salivary gland tumor that bears striking histologic similarity to lobular carcinoma of the breast. While this tumor has an apocrine phenotype that supports classification as a variant of SDC, it infrequently arises in association with conventional SDC. Furthermore, discohesive architecture can be seen in non-apocrine salivary carcinomas, raising the possibility that discohesive growth should define a separate entity. In this study, we aimed to perform comprehensive molecular profiling of SDC-RF to better understand its pathogenesis and classification. We documented the clinicopathologic features of 9 cases of SDC-RF and performed immunostains including AR, GCDFP, and e-cadherin on all cases. We also performed targeted next generation sequencing (NGS) panels on 7 cases that had sufficient tissue available. The SDC-RF represented 8 men and 1 woman with a median age of 67 years (range 63-83 years) and included 6 parotid, 2 buccal, and 1 submandibular primary. All tumors were uniformly composed of discohesive cells with abundant eosinophilic cytoplasm; signet-ring cell features were seen in 2 cases. All tumors were also positive for AR (100%) and GCDFP (100%), and 7 tumors (78%) displayed lost or abnormal e-cadherin. NGS highlighted concomitant PIK3CA and HRAS mutations in 4 tumors, with additional cases harboring TP53, PTEN, and AKT1 mutations. Furthermore, CDH1 alterations were seen in 6 cases, including a novel CDH1::CORO7 fusion. Among 5 patients with follow-up available, 3 (60%) developed local recurrence and widespread distant metastasis and died of disease at a median 20 months (range 10-48 months). Overall, our findings confirm frequent CDH1 mutations and e-cadherin inactivation in SDC-RF, similar to discohesive tumors from other sites. We also highlight an apocrine molecular profile similar to conventional SDC. However, occasional AKT1 mutation and signet-ring features suggest SDC-RF may also be related to mucinous adenocarcinoma. As more salivary tumors with discohesive growth are identified, it may become clearer whether SDC-RF should remain in the SDC family or be recognized as a separate entity.

Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer

Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 (Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.

Abstract PD14-07: Bromodomain and Extra-Terminal motif (BET) inhibitors are a rational therapeutic choice for treatment of invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer accounting for approximately 10-15% of all breast tumours. ILC is characterized by inactivation of E-Cadherin and cancer cells that invade the stroma in a "single-file" pattern. Women with ILC are more likely to have hormone receptor-positive disease. ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries. ILC are currently treated in the same manner as all other ER+ breast cancers. Like invasive ductal carcinoma (IDC), anti-estrogen resistance has emerged as a significant problem in the management of ILC. Approximately half of the recurrences of ER+ breast tumors respond to anti-endocrine treatment, while the other half are resistant. Additionally, anti-estrogen-resistant breast tumor cells appear to have/acquire a more aggressive, invasive phenotype compared to their anti-estrogen-responsive counterparts. ILC is considered to be chemo-resistant and ILC patients receive no additional benefit from adjuvant chemotherapy. As such, there is a pressing need to develop tailored therapeutic options for endocrine-resistant ILC patients. The bromodomain and extra-terminal (BET) motif family are epigenetic readers that bind to acetylated histones, recruiting co-factors to regulate transcription. As part of the FP7 RATHER project aimed at identifying rational treatment options for ILC, we discovered that BRD3 (uniquely among the BET family members) is a marker of poor prognosis in ILC but not in ER+ breast cancers as a whole. We subsequently validated this in an independent cohort from the METABRIC study. These data suggest that Brd3 may play a significant role in tumour progression in ILC and may be a rational therapeutic target for lobular tumours. Using the two ILC cell lines SUM44PE (SUM44) and MDA MB 134 VI (MM134) previously shown to be anti-endocrine resistant we assessed the therapeutic potential of BET inhibition. We found that ILC cell lines that do not respond to anti-endocrine therapy are sensitive to a panel of BET inhibitors in both 2D and 3D assays. In particular the BET inhibitors JQ1 and Mivebresib had the highest potency, these BET inhibitors were selected for further research. Next, a multi-omics approach merging RNA-sequencing with mass spectrometry was utilised to dissect the transcriptional networks employed by BET inhibitors in this ILC setting. RNA-sequencing revealed dysregulated pathways in cell cycle division, DNA damage, apoptosis and MAPK signalling following treatment. Further, rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was carried out to find the binding partners of the BRD proteins. Integrated pathway analysis through Genome Enhancer was used to assess the master regulators (MTRs) which drive BET inhibitor function. There was 142 MTRs found across the two cell lines treated with both inhibitors. Of note the MTR, FGFR3, was shown to regulate the downstream targets of both BET inhibitors. Further analysis of BET inhibition combined with erdafitinib, an FGFR inhibitor, drives increased cell inhibition compared to either agent alone. Finally, the efficacy of JQ1 in targeting ILC was assessed in vivo. We utilised the ILC cell-derived xenograft (CDX) models established by mammary intraductal implantation. The efficacy of BET inhibition alone and in combination with anti-endocrine therapies, tamoxifen and fulvestrant were assessed in the MM134 cell line. We found that JQ1 works synergistically with tamoxifen to significantly decrease tumour burden and metastatic potential in this tamoxifen resistant ILC model. Taken together this data highlights the need for tailored therapeutics in ILC research and highlights the use of BET inhibition in the anti-endocrine resistant ILC setting. Citation Format: Elspeth Ward, Anna Blümel, Emer Conroy, Grainne Cremin, Binbin Gao, William Gallagher, Idalia Cruz, Leena Hilakivi-Clarke, George Sflomos, Cathrin Brisken, Darran O'Connor. Bromodomain and Extra-Terminal motif (BET) inhibitors are a rational therapeutic choice for treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-07.

Upregulation of CD36, a Fatty Acid Translocase, Promotes Colorectal Cancer Metastasis by Increasing MMP28 and Decreasing E-Cadherin Expression.

Simple SummaryColorectal cancer is the second leading cause of cancer-related death in the world. Upregulation of fatty acid metabolism is a hallmark of cancer and recent studies demonstrate that blocking fatty acid uptake is a promising therapeutic strategy. We have previously shown that CD36, a transporter of fatty acid, promotes colorectal cancer tumor growth. We have also demonstrated that high expression of CD36 is associated with cancer cells that are prone to metastasis. Here, in studying the role of CD36 in colorectal cancer, we found that CD36 promotes colorectal cancer invasion in vitro and metastasis in vivo and that overexpression of CD36 upregulates expression of the matrix metalloproteinase MMP28. Our data demonstrates that MMP28 cleaves and decreases expression of E-cadherin, a marker for epithelial-to-mesenchymal transition in colorectal cancers. This newly defined CD36–MMP28–E-cadherin axis provides new therapeutic targets for the treatment of colorectal cancer.Altered fatty acid metabolism continues to be an attractive target for therapeutic intervention in cancer. We previously found that colorectal cancer (CRC) cells with a higher metastatic potential express a higher level of fatty acid translocase (CD36). However, the role of CD36 in CRC metastasis has not been studied. Here, we demonstrate that high expression of CD36 promotes invasion of CRC cells. Consistently, CD36 promoted lung metastasis in the tail vein model and GI metastasis in the cecum injection model. RNA-Seq analysis of CRC cells with altered expression of CD36 revealed an association between high expression of CD36 and upregulation of MMP28, a novel member of the metallopeptidase family of proteins. Using shRNA-mediated knockdown and overexpression of CD36, we confirmed that CD36 regulates MMP28 expression in CRC cells. siRNA-mediated knockdown of MMP28 decreases invasion of CRC cells, suggesting that MMP28 regulates the metastatic properties of cells downstream of CD36. Importantly, high expression of MMP28 leads to a significant decrease in active E-cadherin and an increase in the products of E-cadherin cleavage, CTF1 and CTF2. In summary, upregulation of CD36 expression promotes the metastatic properties of CRC via upregulation of MMP28 and an increase in E-cadherin cleavage, suggesting that targeting the CD36–MMP28 axis may be an effective therapeutic strategy for CRC metastasis.

Abstract LB246: E-cadherin loss drives Id2-dependent dampening of cell cycle progression and predicts increased susceptibility to CDK4/6 inhibition in lobular breast cancer

Abstract Invasive lobular breast carcinoma (ILC) is a low grade and mostly chemo-refractory luminal-type breast cancer that has been linked to sustained proliferative quiescence and long-term latency relapses (15-20 years). Loss of E-cadherin causes metastatic lobular breast cancer, partly through acquisition of anchorage independence. It is however still unknown how ILC cells control the balance between proliferative indolence and cell cycle re-entry at the metastatic site. We show here that E-cadherin loss leads to upregulation of Id2 through p120-catenin/Kaiso-dependent transcriptional derepression. Anchorage independent conditions further exacerbate p120-driven Id2 expression, leading to a sustained G0/G1 cell cycle arrest through binding of cytosolic Id2 to hypo-phosphorylated Rb. Intriguingly, we find that E-cadherin inactivation causes increased sensitivity to CDK4/6 inhibition in mouse and human breast cancer cell lines and primary tumor organoids. Finally, we find that Id2 expression is elevated in human ILC when compared to ductal breast cancers. Based on these data, we propose that combined E-cadherin loss and cytosolic Id2 expression can be used for the differential diagnosis of ILC and represent a candidate predictive biomarker pair for cell cycle targeting drug efficacy. Citation Format: Max Antonius Klaus Rätze, Thijs Koorman, Thijmen Sijnesael, Blessing Bassey-Archibong, Robert van de Ven, Lotte Enserink, Daan Visser, Sridevi Jaksani, Elvira Bakker, François Richard, Andrew Tutt, Rebecca Steele, Stephen Pettitt, Christopher J. Lord, Amanda Fitzpatrick, Clare Isacke, Paul J. van Diest, Christine Desmedt, Juliet M. Daniel, Patrick W.B. Derksen. E-cadherin loss drives Id2-dependent dampening of cell cycle progression and predicts increased susceptibility to CDK4/6 inhibition in lobular breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB246.

Trop-2 inactivates E-cadherin and drives colon cancer metastasis.

e15576 Background: Metastatic diffusion is the biggest hurdle for colon cancer (CRC) cure and the identification of decisive drivers of CRC metastasis is an urgent need in CRC care. Methods: The expression of target molecules in primary tumors and metastases was systematically assessed by DNA array and IHC analysis. Cell-cell adhesion capacity was quantified in 2D cell cultures and in HCT116 CRC cell spheroids. Pro-metastatic impact of wtTrop-2 and activated, tail-less Trop-2 (Δcyto) was assessed in vivo in orthotopic diffusion models of KM12SM CRC cells. Primary CRC and metastasis transcriptomes were analyzed for differential induction of EMT determinants. Kaplan–Meier plots were used to illustrate survival and metastatic relapse in independent case series of CRC patients. Results: Trop-2 was identified as uniquely upregulated in CRC models by transcriptome analysis. wtTrop-2 and ΔcytoTrop-2 were shown to induce cell migration, wound-healing and resistance to apoptosis induction . wtTrop-2 increased the metastatic capacity of KM12SM cells, raising metastasis diffusion from 45% for control cells to 90% for wtTrop-2 transfectants. The constitutively-active ΔcytoTrop-2 further boosted metastatic spreading, with metastatic livers reaching up to four times their normal size. Constitutive high expression of E-cadherin was revealed in cancer metastases. No evidence was obtained for transcriptional down-regulation of epithelial differentiation biomarkers. No induction of EMT transcription factors was observed in Trop-2-activated cells. Trop-2 tightly bound E-cadherin and caused its release from the cytoskeleton, for loss of cell-cell adhesion and activation of β-catenin. The Trop-2/E-cadherin/β-catenin-driven pro-metastatic program was recapitulated in CRC patients and was shown to impact on CRC metastatic relapse and overall patient survival. Conclusions: We identify Trop-2-driven functional inactivation of E-cadherin as a key driver of metastatic diffusion in CRC. These findings may pave the way for novel multi-marker personalized diagnostics and anti-cancer therapies. [Table: see text]

101P E-cadherin inactivation by Trop-2 drives EMT-less metastatic relapse in triple-negative breast cancer

101P E-cadherin inactivation by Trop-2 drives EMT-less metastatic relapse in triple-negative breast cancer

Abstract PD15-04: Trop-2 inactivation of E-cadherin drives triple negative breast cancer relapse

Abstract Hundreds of proteins/genes have been linked to the metastatic phenotype. However, consistent markers of tumour aggressiveness and metastatic potential in breast cancer patients were not identified, not even including proteomic analysis and large-scale genome sequencing.Metastasis-associated genes were predicted to include not only drivers of the metastatic phenotype, but also secondary events, together with adaptive, counterbalancing changes. Thus, to identify candidates with a required role in metastatic diffusion, we looked for genes that were concordantly dysregulated across orthogonal cancer metastasis settings. This led us to identify Trop-2 as uniquely upregulated and associated to metastasis in experimental models of breast cancer, as well as in other solid tumors. We identified functional inactivation of E-cadherin by Trop-2 as the main motor of metastatic diffusion of such metastatic systems. Trop-2 binding to E-cadherin inactivated its cell-cell adhesion function, through release from the cytoskeleton, for activation of β-catenin transcriptional activity. This led to anti-apoptotic signaling, increased cell migration capacity and enhanced cancer cell survival. We showed that this mechanism led to metastatic diffusion of xenotransplants growing in immunosuppressed mice. An E-cadherin-inactivation metastasis program was then shown to be recapitulated in breast cancer patients, as well as in other solid tumors, over 24 independent case series, encompassing 13,042 primary tumours. Aggressive triple-negative breast cancers were shown to be driven toward global relapse by Trop-2 overexpression, through E-cadherin inactivation and activation of β-catenin transcriptional activity. No disease recurrence was observed in control cases over +12 years of follow-up. These finding lead to a novel paradigm of a Trop-2-driven, E-cadherin-inactivation program as a main metastasis driver in solid tumors. This may open far-reaching perspectives in diagnostic procedures and anti-cancer therapies. Citation Format: Saverio Alberti, Marco Trerotola, Valeria Relli, Rossano Lattanzio, Martina Ceci, Khouloud Boujnah, Valeria Garbo, Antonino Moschella, Patrizia Querzoli, Massimo Pedriali, Laura Antolini, Emanuela Guerra. Trop-2 inactivation of E-cadherin drives triple negative breast cancer relapse [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-04.

Inactivation of E-cadherin by Trop-2 drives colon cancer metastasis.

105 Background: Tumor metastasis is the main cause of death of colon cancer patients and the biggest hurdle for cancer cure. We set to identify decisive drivers and of pivotal therapy targets for colon cancer metastasis. Methods: IHC analysis quantified the expression of target molecules in primary tumors and metastases. Cell-cell adhesion capacity was assessed in vitro and in HCT116 colon cancer cell spheroids. Pre-clinical models of orthotopic growth of KM12SM colon cancer cells and metastatic diffusion to the liver were utilized to assess metastatic spreading force of wtTrop-2 and of the constitutively-active, tail-less form of Trop-2 (Δcyto). Xenotransplant and metastasis transcriptomes were analyzed for differential induction of EMT determinants. Kaplan–Meier plots were used to illustrate survival and metastatic relapse in independent case series of colon cancer patients. Results: wtTrop-2 was shown to induce wound-healing. ΔcytoTrop-2 further increased cell migration ability. Both wtTrop-2 and ΔcytoTrop-2 induced resistance to apoptosis in vitro and in vivo. wtTrop-2 strikingly increased the metastatic capacity of KM12SM cells, raising metastasis rates from 45% for control cells to 90% for wtTrop-2 transfectants. The constitutively-active ΔcytoTrop-2 further boosted metastatic spreading, with metastatic livers reaching up to four times their normal size. Cancer metastases revealed high levels of E-cadherin, in the absence of transcriptional down-regulation. EMT transcription factors were largely missing from Trop-2-activated cells. Rather, binding to Trop-2 was shown to cause the release of E-cadherin from the cytoskeleton, loss of cell-cell adhesion and activation of β-catenin. This global, Trop-2/E-cadherin/β-catenin-driven pro-metastatic program was recapitulated in colon cancer patients and was shown to impact on colon cancer metastatic relapse and overall patient survival. Conclusions: We identify Trop-2-driven functional inactivation of E-cadherin as a widespread driver of metastatic diffusion in colon cancer, opening novel avenues for personalized diagnostic procedures and anti-cancer therapies. [Table: see text]

57P Functional inactivation of E-cadherin by Trop-2 drives cancer metastasis

57P Functional inactivation of E-cadherin by Trop-2 drives cancer metastasis

Abstract P3-10-06: Combination of BET and FGFR inhibition as a rational therapeutic strategy for invasive lobular breast cancer

Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor-positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 (uniquely among BRD family members) was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression (and not other BRD members) was associated with poor recurrence-free survival (p&amp;lt;0.01, HR=3.16, CI 1.24-8.03). Using a two ILC cell lines (SUM44PE and MDA-MB134VI) we found that ILC cells were relatively resistant to the anti-estrogen therapies tamoxifen and fulvestrant compared to those derived from IDC. Next, we tested whether the ILC cell lines were sensitive to BET protein inhibition using the pan-BET family inhibitor JQ1. Interestingly, while JQ1 inhibited cell growth in both ILC cell lines tested, apoptosis was only induced in SUM44PE cells, while MDA-MB134VI cells exhibited G1 arrest. Dynamic BH3 profiling was used to dissect the underlying anti-apoptotic dependencies in each ILC cell type and showed that in the JQ1-resistant MDA-MB134VI cells, survival was dependent on sustained Bcl-Xl expression and transcriptional rewiring inducing Fibroblast Growth Factor Receptor 1 (FGFR1). This increase in FGFR1 was also evident in invasive ductal carcinoma (IDC) cell lines that also did not respond to JQ1. The combination of JQ1 and FGFR1 inhibitor, or JQ1 and the Bcl2/Bcl-Xl/Bcl-W inhibitor navitoclax (ABT-263), were highly effective at inhibiting growth in both 2D and 3D models of ILC and IDC. Interestingly, we found in the RATHER cohort of 61 ILC patients that 20% had FGFR1 amplification suggesting that this therapeutic approach may have utility in a large number of ILC patients. Conclusions: We provide evidence that BETi either alone or in combination with FGFR1 inhibitors or BH3 mimetics may be a useful therapeutic strategy for recurrent ILC patients. Citation Format: Darran P O'Connor, Louise Walsh, Kathryn Haley, Bruce Moran, Brian Mooney, Stephen Madden, Sudipto Das, Oscar Rueda, Catriona Dowling, Damir Vareslija, Suet-Feung Chin, Sabine Linn, Leonie Young, Karin Jirstrom, John P Crown, Rene Bernards, Carlos Caldas, William M Gallagher, Triona Ni Chonghaile. Combination of BET and FGFR inhibition as a rational therapeutic strategy for invasive lobular breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-06.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Characterizing the invasion of different breast cancer cell lines with distinct E-cadherin status in 3D using a microfluidic system

E-cadherin is a cell-cell adhesion protein that plays a prominent role in cancer invasion. Inactivation of E-cadherin in breast cancer can arise from gene promoter hypermethylation or genetic mutation. Depending on their E-cadherin status, breast cancer cells adopt different morphologies with distinct invasion modes. The tumor microenvironment (TME) can also affect the cell morphology and invasion mode. In this paper, we used a previously developed microfluidic system to quantify the three-dimensional invasion of breast cancer cells with different E-cadherin status, namely MCF-7, CAMA-1 and MDA-MB-231 with wild type, mutated and promoter hypermethylated E-cadherin, respectively. The cells migrated into a stable and reproducible microfibrous polycaprolactone mesh in the chip under a programmed stable chemotactic gradient. We observed that the MDA-MB-231 cells invaded the most, as single cells. MCF-7 cells collectively invaded into the matrix more than CAMA-1 cells, maintaining their E-cadherin expression. The CAMA-1 cells exhibited multicellular multifocal infiltration into the matrix. These results are consistent with what is seen in vivo in the cancer biology literature. In addition, comparison between complete serum and serum gradient conditions showed that the MDA-MB-231 cells invaded more under the serum gradient after one day, however this behavior was inverted after 3 days. The results showcase that the microfluidic system can be used to quantitatively assess the invasion behavior of cancer cells with different E-cadherin expression, for a longer period than conventional invasion models. In the future, it can be used to quantitatively investigate effects of matrix structure and cell treatments on cancer invasion.

Rebalancing of actomyosin contractility enables mammary tumor formation upon loss of E-cadherin

E-cadherin (CDH1) is a master regulator of epithelial cell adherence junctions and a well-established tumor suppressor in Invasive Lobular Carcinoma (ILC). Intriguingly, somatic inactivation of E-cadherin alone in mouse mammary epithelial cells (MMECs) is insufficient to induce tumor formation. Here we show that E-cadherin loss induces extrusion of luminal MMECs to the basal lamina. Remarkably, E-cadherin-deficient MMECs can breach the basal lamina but do not disseminate into the surrounding fat pad. Basal lamina components laminin and collagen IV supported adhesion and survival of E-cadherin-deficient MMECs while collagen I, the principle component of the mammary stromal micro-environment did not. We uncovered that relaxation of actomyosin contractility mediates adhesion and survival of E-cadherin-deficient MMECs on collagen I, thereby allowing ILC development. Together, these findings unmask the direct consequences of E-cadherin inactivation in the mammary gland and identify aberrant actomyosin contractility as a critical barrier to ILC formation.