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Intratumoral heterogeneity has been described for various tumor types and models of human cancer, and can have profound effects on tumor progression and drug resistance. This study describes an in-depth analysis of molecular and functional heterogeneity among subclonal populations (SCPs) derived from a single triple-negative breast cancer cell line, including copy number analysis, whole-exome and RNA sequencing, proteome analysis, and barcode analysis of clonal dynamics, as well as functional assays. The SCPs were found to have multiple unique genetic alterations and displayed significant variation in anchorage independent growth and tumor forming ability. Analyses of clonal dynamics in SCP mixtures using DNA barcode technology revealed selection for distinct clonal populations in different in vitro and in vivo environmental contexts, demonstrating that in vitro propagation of cancer cell lines using different culture conditions can contribute to the establishment of unique strains. These analyses also revealed strong enrichment of a single SCP during the development of xenograft tumors in immune-compromised mice. This SCP displayed attenuated interferon signaling in vivo and reduced sensitivity to the antiproliferative effects of type I interferons. Reduction in interferon signaling was found to provide a selective advantage within the xenograft microenvironment specifically. In concordance with the previously described role of interferon signaling as tumor suppressor, these findings suggest that similar selective pressures may be operative in human cancer and patient-derived xenograft models.

PD-1 blockade is highly effective in classical Hodgkin lymphomas (cHLs), which exhibit frequent copy-number gains of CD274 (PD-L1) and PDC1LG2 (PD-L2) on chromosome 9p24.1. However, in this largely MHC-class-I-negative tumor, the mechanism of action of anti-PD-1 therapy remains undefined. We utilized the complementary approaches of T cell receptor (TCR) sequencing and cytometry by time-of-flight analysis to obtain a peripheral immune signature of responsiveness to PD-1 blockade in 56 patients treated in the CheckMate 205 phase II clinical trial (NCT02181738). Anti-PD-1 therapy was most effective in patients with a diverse baseline TCR repertoire and an associated expansion of singleton clones during treatment. CD4+, but not CD8+, TCR diversity significantly increased during therapy, most strikingly in patients who had achieved complete responses. Additionally, patients who responded to therapy had an increased abundance of activated natural killer cells and a newly identified CD3−CD68+CD4+GrB+ subset. These studies highlight the roles of recently expanded, clonally diverse CD4+ T cells and innate effectors in the efficacy of PD-1 blockade in cHL. In a phase II clinical trial of patients with classical Hodgkin lymphoma, peripheral CD4+ T cell receptor diversity and the abundance of mature natural killer cells and CD3−CD68+CD4+GrB+ innate cells were associated with favorable responses to anti-PD-1 monotherapy.

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk., Organoid technology has enabled the generation of several breast cancer organoids. Here, the authors combine propagation of normal human mammary tissues with mass cytometry to evaluate the ability of organoid culture technologies to preserve stem cells and differentiated cell types.

To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA-Exchange-Imaging (DEI), for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DNA-Exchange-Imaging allows us to apply it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ~300 nm down to sub-20-nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

Fusions involving the neuregulin 1 gene (NRG1) occur at low frequency in pancreatic, lung, and other cancers. NRG1 fusion oncoproteins bind to HER3, leading to heterodimerization with HER2 and potent activation of downstream signaling mainly via the PI3K-AKT pathway. Zenocutuzumab (Zeno, MCLA-128), an ADCC-enhanced anti-HER2×HER3 bi-specific antibody, uniquely ‘docks' on HER2, to position the antibody and subsequently ‘block' NRG1 from interacting with HER3, effectively preventing HER2:HER3 heterodimerization and downstream signaling. Our goal in this study was to evaluate the efficacy of Zeno in preclinical models of NRG1 fusion-positive cancers. We tested Zeno in a panel of isogenic and patient-derived cell line and xenograft (PDX) models of lung, breast and pancreatic cancers. Cell lines either expressed an NRG1 fusion endogenously (MDA-MB-175-VII, DOC4-NRG1) or by lentiviral transfer of cDNAs (ATP1B1-NRG1 and SLC3A2-NRG1 in H6c7 pancreatic ductal cell line; CD74-NRG1 and VAMP2-NRG1 in immortalized human bronchial epithelial cells; and DOC4-NRG1 in MCF7 breast cancer cells). PDX models were generated from NSCLC samples harboring CD74-NRG1 (ST3204) or SLC3A2-NRG1 (LUAD-0061AS3) fusions and from a high grade serous ovarian cancer harboring a CLU-NRG1 fusion (OV-10-0050). Zeno treatment of NRG1 fusion-expressing breast, pancreatic, and lung cancer cell lines resulted in dose-dependent reduction of growth and abrogated phosphorylation of HER3, HER4, AKT, p70S6 kinase and STAT3 in all cell lines tested. Phosphorylation of HER2, EGFR and MEK/ERK was inhibited, albeit with some variation, in a cell line-specific manner. Growth of isogenic control cell lines without NRG1 fusion was not significantly altered. In breast and lung cancer cell lines, Zeno treatment down-regulated cyclin D1 expression and induced expression of the negative cell cycle regulators P21 or P27. Evidence of apoptosis activation (cleaved PARP, expression of BIM and PUMA) was also observed in cells exposed to Zeno. Treatment of mice bearing LUAD-0061AS3, ST3204 and OV-10-0050 PDX tumors (2.5, 8, 25 mg/kg, QW) caused a dose-dependent inhibition of tumor growth, with tumor shrinkage observed at higher doses. Finally, we assessed the ability of Zeno to induce antibody-dependent cellular cytotoxicity using a chromium release assay and peripheral blood mononuclear cells. Zeno induced significant cytotoxicity in MDA-MB-175-VII cells while a non-ADCC enhanced, non-specific IgG had no effect. Here we show that Zeno effectively blocks the growth of NRG1 fusion-positive cell line and xenograft models of tumors arising from lung, pancreas and other organs, and these results support the continued development of Zeno to treat patients with this molecularly defined subset of cancers. Citation Format: Igor Odintsov, Inna Khodos, Madelyn Espinosa-Cotton, Allan J. Lui, Marissa Mattar, Alison M. Schram, Ron C. Schackmann, Jeroen Lammerts van Bueren, Cecile A. Geuijen, Elisa de Stanchina, Marc Ladanyi, Romel Somwar. The HER2×HER3 bi-specific antibody Zenocutuzumab is effective at blocking growth of tumors driven by NRG1 gene fusions [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 956.

Background: Mutations in BRCA1 and BRCA2 are associated with a high risk of developing breast cancer, but the earliest molecular changes that lead to the transformation of mammary cells in the setting of BRCA1/2 heterozygosity remain unknown. We previously demonstrated that breast organoid cultures derived from histologically normal tissues can preserve all of the major mammary epithelial lineages for further study in vitro1. In addition, organoid culture medium is fully defined, enabling modulation of the medium to enhance the growth of distinct mammary subpopulations. Here, we undertook the development of a large biobank of breast organoids derived from patients with and without increased breast cancer risk, including patients with inherited mutations in BRCA1 and BRCA2. Methods: Breast samples with normal histology (taken from reduction mammoplasties or prophylactic mastectomies), were digested for ~2 hours using collagenase, embedded in basement membrane extract, and grown in a fully-defined organoid medium1. CyTOF profiling was performed using our previously published mammary-specific heavy metal-tagged antibody panel1. Lentiviral transduction was performed to knock-down BRCA1 and/or overexpress mutant p53. Murine engraftment of normal breast organoids was performed by intra-ductal injection of ~1,000 organoids into NCG mice using a modification of protocols for breast cancer cell lines2, with removal of mammary glands after 3 months for detailed immunohistochemistry analysis. Results: A living biobank of > 100 normal breast organoids derived from patients with and without inherited mutations in breast cancer predisposition genes, including BRCA1 and BRCA2, was established. Cultures were generated with high efficiency (>95%) and could be serially passaged with the longest cultures > 16 months. CyTOF profiling of organoids revealed the maintenance of multiple epithelial cell subtypes, with at least one subtype of luminal cells enriched in tissues and organoids from BRCA1/2 mutation carriers. Protein and RNA expression patterns of breast organoids were found to correlate with patient tissues analyzed using a combination of single-cell analyses (CyTOF, single-cell RNA sequencing, and immunohistochemistry). The impact of factors present in the organoid medium on distinct mammary epithelial cell subtypes was assessed by CyTOF, enabling identification of conditions that promote expansion of cell populations that are enriched in tissues from BRCA1/2 mutation carriers. Furthermore, normal breast cell types could be engrafted into the murine mammary gland, and could be modified by lentiviral transduction for gene transfer, enabling future studies of the tumorigenic potential of distinct normal and premalignant epithelial cell subtypes. Conclusion: We have shown that organoid cultures can be used to propagate normal breast tissues with high efficiency, preserve normal as well as potential premalignant breast epithelial cell types, and model methods to inhibit precancerous cells in vitro. Thus, organoids are a useful complement to murine and other models of breast cancer development, and can ultimately be used to identify potential cancer interception strategies for patients at high risk of developing breast cancer. References:1.Rosenbluth JM, Schackmann RCJ, Gray GK, et al: Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages. Nat Commun 11:1711, 20202.Zoeller JJ, Bronson RT, Selfors LM, et al: Niche-localized tumor cells are protected from HER2-targeted therapy via upregulation of an anti-apoptotic program in vivo. NPJ Breast Cancer 3:18, 2017 Citation Format: Jennifer M Rosenbluth, Carman Man-Chung Li, Kenneth G Gray, Walter Gao, Ron CJ Schackmann, Jason J Zoeller, Laura M Selfors, Kaitlin Moore, Deborah Dillon, Judy Garber, Joan S Brugge. A living biobank of normal mammary organoids derived from patients at low and increased risk of developing breast cancer [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 PD7-03.

In classical Hodgkin lymphoma (cHL), the host antitumor immune response is ineffective. Hodgkin Reed-Sternberg (HRS) cells have multifaceted mechanisms to evade the immune system, including 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) genetic alterations, overexpression of PD-1 ligands, and associated T-cell exhaustion and additional structural bases of aberrant antigen presentation. The clinical success of PD-1 blockade in cHL suggests that the tumor microenvironment (TME) contains reversibly exhausted T effector cells (Teffs). However, durable responses are observed in patients with β2-microglobulin/major histocompatibility complex (MHC) class I loss on HRS cells, raising the possibility of non-CD8(+) T cell–mediated mechanisms of efficacy of PD-1 blockade. These observations highlight the need for a detailed analysis of the cHL TME. Using a customized time-of-flight mass cytometry panel, we simultaneously assessed cell suspensions from diagnostic cHL biopsies and control reactive lymph node/tonsil (RLNT) samples. Precise phenotyping of immune cell subsets revealed salient differences between cHLs and RLNTs. The TME in cHL is CD4(+) T-cell rich, with frequent loss of MHC class I expression on HRS cells. In cHLs, we found concomitant expansion of T helper 1 (Th1)-polarized Teffs and regulatory T cells (Tregs). The cHL Th1 Tregs expressed little or no PD-1, whereas the Th1 Teffs were PD-1(+). The differential PD-1 expression and likely functional Th1-polarized CD4(+) Tregs and exhausted Teffs may represent complementary mechanisms of immunosuppression in cHL.

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 °C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

Summary The epithelial adherens junction is an E-cadherin-based complex that controls tissue integrity and is stabilized at the plasma membrane by p120-catenin (p120, also known as CTNND1). Mutational and epigenetic inactivation of E-cadherin has been strongly implicated in the development and progression of cancer. In this setting, p120 translocates to the cytosol where it exerts oncogenic properties through aberrant regulation of Rho GTPases, growth factor receptor signaling and derepression of Kaiso (also known as ZBTB33) target genes. In contrast, indirect inactivation of the adherens junction through conditional knockout of p120 in mice was recently linked to tumor formation, indicating that p120 can also function as a tumor suppressor. Supporting these opposing functions are findings in human cancer, which show that either loss or cytoplasmic localization of p120 is a common feature in the progression of several types of carcinoma. Underlying this dual biological phenomenon might be the context-dependent regulation of Rho GTPases in the cytosol and the derepression of Kaiso target genes. Here, we discuss past and present findings that implicate p120 in the regulation of cancer progression and highlight opportunities for clinical intervention.

Metastatic breast cancer remains the chief cause of cancer-related death among women in the Western world. Although loss of cell–cell adhesion is key to breast cancer progression, little is known about the underlying mechanisms that drive tumor invasion and metastasis. Here, we show that somatic loss of p120-catenin (p120) in a conditional mouse model of noninvasive mammary carcinoma results in formation of stromal-dense tumors that resemble human metaplastic breast cancer and metastasize to lungs and lymph nodes. Loss of p120 in anchorage-dependent breast cancer cell lines strongly promoted anoikis resistance through hypersensitization of growth factor receptor (GFR) signaling. Interestingly, p120 deletion also induced secretion of inflammatory cytokines, a feature that likely underlies the formation of the prometastatic microenvironment in p120-negative mammary carcinomas. Our results establish a preclinical platform to develop tailored intervention regimens that target GFR signals to treat p120-negative metastatic breast cancers. Cancer Res; 73(15); 4937–49. ©2013 AACR.

YAP is a transcriptional coactivator that controls organ expansion and differentiation and is inhibited by the Hippo pathway in cells in interphase. Here, we demonstrated that, during mitosis, YAP localized to the midbody and spindle, subcellular structures that are involved in cytokinesis, the process by which contraction of the cytoskeleton produces two daughter cells. Furthermore, YAP was phosphorylated by CDK1, a kinase that promotes cell cycle progression. Knockdown of YAP by shRNA or expression of a nonphosphorylatable form of YAP delayed the separation of daughter cells (called abscission) and induced a cytokinesis phenotype associated with increased contractile force, membrane blebbing and bulges, and abnormal spindle orientation. Consequently, these defects led to an increased frequency of multinucleation, micronuclei, and aneuploidy. YAP was required for proper localization of proteins that regulate contraction during cytokinesis, including ECT2, MgcRacGap, Anillin, and RHOA. In addition, depletion of YAP increased the phosphorylation of myosin light chain, which would be expected to activate the contractile activity of myosin II, the molecular motor involved in cytokinesis. The polarity scaffold protein PATJ coprecipitated with YAP and colocalized with YAP at the cytokinesis midbody, and knockdown of PATJ phenocopied the cytokinetic defects and spindle orientation alterations induced by either YAP depletion or expression of a nonphosphorylatable YAP mutant. Together, these results reveal an unanticipated role for YAP in the proper organization of the cytokinesis machinery during mitosis through interaction with the polarity protein PATJ.

Metastatic breast cancer is the major cause of cancer-related death among women in the Western world. Invasive carcinoma cells are able to counteract apoptotic signals in the absence of anchorage, enabling cell survival during invasion and dissemination. Although loss of E-cadherin is a cardinal event in the development and progression of invasive lobular carcinoma (ILC), little is known about the underlying mechanisms that govern these processes. Using a mouse model of human ILC, we show here that cytosolic p120-catenin (p120) regulates tumor growth upon loss of E-cadherin through the induction of anoikis resistance. p120 conferred anchorage independence by indirect activation of Rho/Rock signaling through interaction and inhibition of myosin phosphatase Rho-interacting protein (Mrip), an antagonist of Rho/Rock function. Consistent with these data, primary human ILC samples expressed hallmarks of active Rock signaling, and Rock controlled the anoikis resistance of human ILC cells. Thus, we have linked loss of E-cadherin - an initiating event in ILC development - to Rho/Rock-mediated control of anchorage-independent survival. Because activation of Rho and Rock are strongly linked to cancer progression and are susceptible to pharmacological inhibition, these insights may have clinical implications for the development of tailor-made intervention strategies to better treat invasive and metastatic lobular breast cancer.

Background: In classical Hodgkin lymphoma (cHL), the rare malignant Hodgkin Reed-Sternberg (HRS) cells are surrounded by an inflammatory infiltrate. Yet, the host anti-tumor immune response is ineffective. HRS cells have multifaceted mechanisms to evade the immune system including 9p24.1/PD-L1/PD-L2 genetic alterations leading to overexpression of PD-1 ligands and subsequent T cell exhaustion, aberrant antigen presentation and modulation of the tumor microenvironment (TME). The clinical success of PD-1 blockade in cHL suggests the TME contains reversibly exhausted T-effectors (Teff). Paradoxically, durable responses are observed in patients with β2M/MHC class I loss on HRS cells, raising the possibility of non-CD8+ mediated mechanisms of efficacy of PD-1 blockade. For this reason, we sought to characterize HRS cells and the surrounding TME. Methods: Using CyTOF technology, we evaluated 7 primary cHL suspensions and 10 reactive lymphoid tissue (RLT) samples at the single-cell protein level. We designed a custom panel of 39 isotope-conjugated antibodies. A combination of surface and intracellular markers distinguish T cell subsets according to lineage, differentiation, polarization, activation and exhaustion. Additional markers were incorporated to identify B cells, NK cells and macrophages. HRS cells were defined by CD15/CD30/Pax5 positivity. Inclusion of β2M and MHC class I allowed assessment of antigen presentation on HRS cells. The data was acquired on a Helios CyTOF and analyzed using a fast k-weighted nearest neighbor algorithm, X-shift. X-shift clustered cells with phenotypic similarities together. Then, samples were separated into cHL and RLT and the contribution of a sample to a given cluster was quantified. Results: Comparison of viable cell suspensions from RLT and cHL revealed loss of naïve T-cells and skewing towards differentiation of Teff in both CD4+ and CD8+ subsets in cHLs. This prompted a second X-shift analysis focused on CD3+ cells, which highlighted salient differences between cHL and RLT within the CD4+ subset. In cHL, we found expansion of Teff and regulatory T cells (Treg) with a reduction of follicular helper T cells. Furthermore, both Treg and Teff populations were largely Th1 (T-bet+/CCR5+) polarized. Evaluation of PD-1 expression showed Tregs had little/no PD-1 while Teff had intermediate/high expression. Hence, Tregs retain functionality in contrast to Teff, which are exhausted, providing two mechanisms of immunosuppression. Manual gating identified HRS cells with a characteristic phenotype: CD15, CD30, Pax5, rosetted by CD4+ T cells. Importantly, we found loss or decrease of β2M and MHC class I in 5/7 cases. Conclusions: The TME in cHL is CD4+ T cell rich with frequent loss of MHC class I on HRS cells. Differential PD-1 expression results in functional CD4+ Tregs and exhausted Teff, a synergistic bases for the observed immunosuppression in cHL. Citation Format: Fathima Z. Cader, Ron C. Schackmann, Xihao Hu, Kirsty Wienand, Robert A. Redd, Bjoern Chapuy, Jing Ouyang, Nicole E. Paul, Evisa Gjini, Mikel Lipschitz, Laura M. Selfors, Philippe Armand, David Wu, Jonathan R. Fromm, Donna Neuberg, Xiaole S. Liu, Scott J. Rodig, Margaret A. Shipp. Single-cell mass cytometry of classical Hodgkin lymphoma defines an exhausted and immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5675.

E-cadherin inactivation underpins the progression of invasive lobular breast carcinoma (ILC). In ILC, p120-catenin (p120) translocates to the cytosol where it controls anchorage independence through the Rho-Rock signaling pathway, a key mechanism driving tumor growth and metastasis. We now demonstrate that anchorage-independent ILC cells show an increase in nuclear p120, which results in relief of transcriptional repression by Kaiso. To identify the Kaiso target genes that control anchorage independence we performed genome-wide mRNA profiling on anoikis resistant mouse ILC cells, and identified 29 candidate target genes including the established Kaiso target Wnt11. Our data indicate that anchorage-independent upregulation of Wnt11 in ILC cells is controlled by nuclear p120 through inhibition of Kaiso-mediated transcriptional repression. Finally, we show that Wnt11 promotes activation of RhoA, which causes ILC anoikis resistance. Our findings thereby establish a mechanistic link between E-cadherin loss and subsequent control of Rho-driven anoikis resistance through p120/Kaiso-dependent expression of Wnt11.

Background & Aims Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential. Methods Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor–related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach. Results CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras–dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras–driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras–proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras–deficient cells against apoptosis. Conclusions Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1–driven metastasis formation.

While it is well established that mutational inactivation of E-cadherin is a causal event in the development and progression of invasive breast cancer, still little is known about how this event controls the metastatic process. Using conditional E-cadherin knockout mouse models of human invasive lobular carcinoma (ILC), we recently demonstrated that p120-catenin (p120) translocates to the cytosol upon E-cadherin inactivation. Here, it acts as an oncogene by regulating Rock-dependent control of tumor growth and metastasis through binding and inhibition of the Rho antagonist Mrip. Furthermore, new evidence indicates that p120 shuttles to the nucleus where it relieves Kaiso-dependent transcriptional repression of Wnt11, leading to autocrine activation of RhoA. Conversely, since p120 controls E-cadherin stability and turnover, we also addressed the hypothesis that p120 may function as a tumor suppressor in breast cancer. In contrast to E-cadherin loss and the development of metastatic ILC, we show that somatic inactivation of p120 results in the formation of stromal-dense tumors resembling human metaplastic carcinoma that metastasize to lungs and lymph nodes. Mechanistically, we demonstrate that acquisition of metastatic potential was acquired through relieve of E-cadherin-dependent repression of growth factor receptor (GFR) function, leading to hyper-activation of GFR signaling and subsequent anchorage independence. In summary, our data show that p120 can act as an oncogene or metastasis suppressor, depending on context and timing of junctional inactivation. Furthermore, our experiments indicate that targeted intervention strategies using Rock inhibitors may be applicable for the treatment of ILC. Based on our findings we also anticipate that patients suffering from E-cadherin or p120 negative breast cancer will benefit from clinically established inhibitors targeting GFR/PI3K/AKT/FOXO pathways in the absence of GFR or PIK3CA activating mutations. Citation Format: Ron CJ Schackmann, Sjoerd Klarenbeek, Miranda van Amersfoort, Jos Jonkers, Paul van Diest, Patrick Derksen. Context-dependent regulation of breast cancer metastasis by E-cadherin and p120-catenin. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A059.