Driver Of Metastatic Progression Research Articles

IFNγ-induced stem-like state of cancer cells as a driver of metastatic progression following immunotherapy

Despite the remarkable success of immune checkpoint blockade (ICB) therapy, most cancer patients still do not respond. We now find that immunotherapy can induce stem-like properties in tumors. Using mouse models of breast cancer, we observe that cancer stem cells (CSCs) show not only enhanced resistance to Tcell cytotoxicity, but that interferon gamma (IFNγ) produced by activated Tcells directly converts non-CSCs to CSCs. IFNγ enhances several CSC phenotypes, such as resistance to chemo- and radiotherapy and metastasis formation. We identified the branched-chain amino acid aminotransaminase 1 (BCAT1) as a downstream mediator of IFNγ-induced CSC plasticity. Targeting BCAT1 invivo improved cancer vaccination and ICB therapy by preventing IFNγ-induced metastasis formation. Breast cancer patients treated with ICB exhibited a similar increase in CSC markers expression indicating comparable responses to immune activation in humans. Collectively, we discover an unexpected, pro-tumoral role for IFNγ that may contribute to cancer immunotherapy failure.

Distinct populations of tumor associated macrophages in colorectal liver metastasis

Background: Most colorectal cancer (CRC) related mortality is due to metastatic disease and colorectal liver metastases (CRLM) are the most common site of metastases. Despite the high incidence of CRLM, little is known about the role of tumor microenvironment (TME) as a driver of metastatic progression. Here in, we performed an in-depth analysis of the transcriptional landscape of CRC metastases using single cell RNA sequencing (scRNAseq), to evaluate the functional heterogeneity and phenotypic diversity of tumor associated macrophages (TAMs) in CRC metastases.

Antizyme Inhibitor 1 Regulates Matrikine Expression and Enhances the Metastatic Potential of Aggressive Primary Prostate Cancer.

AZIN1 was identified as driver of metastatic progression in high-risk prostate cancer through matrikine regulation.

Zinc transporter SLC39A13/ZIP13 facilitates the metastasis of human ovarian cancer cells via activating Src/FAK signaling pathway

BackgroundZinc transporters have been found to be associated with the pathogenesis of numerous human diseases including cancer. As the most lethal gynecologic malignancy, ovarian cancer is characterized by rapid progression and widespread metastases. However, the function and underlying mechanism of zinc transporters in ovarian cancer metastasis remain unclear.MethodsThe relationship between zinc transporter gene expressions and clinical outcomes of ovarian cancer was assessed with the online database Kaplan-Meier plotter (http://kmplot.com/analysis/). Immunohistochemistry was performed to investigate the prognostic importance of ZIP13. The expression of ZIP13 in ovarian cancer cell lines was depleted to explore its effect on proliferation, adhesion, migration, and invasion both in vitro and in vivo assays. RNA-Seq, quantitative RT-PCR, and western blot analysis were performed to explore ZIP13-regulated downstream target genes.ResultsThe expressions of several zinc transporters were highly associated the clinical outcomes of ovarian cancer patients. Among them, high ZIP13 expression was an independent prognostic factor for poor survival in patients with ovarian cancer. ZIP13 knockout suppressed the malignant phenotypes of ovarian cancer cells both in vitro and in vivo. Further investigation revealed that ZIP13 regulated intracellular zinc distribution and then affected the expressions of genes involved in extracellular matrix organization and cytokine-mediated signaling pathway. This led to the activation of Src/FAK pathway with increased expressions of pro-metastatic genes but decreased expressions of tumor suppressor genes.ConclusionsZIP13 is shown to be a novel driver of metastatic progression by modulating the Src/FAK signaling pathway, which may serve as a promising biomarker for prognostic evaluation and targeted therapy in ovarian cancer.

Abstract PS19-01: A sense-antisense RNA interaction drives metabolic reprogramming in metastatic breast cancer

Abstract Background: Metabolic reprogramming is a hallmark of breast cancer progression. However, the underlying regulatory pathways that initiate and maintain this process remain largely unexplored. Recently, we have identified a novel antisense RNA that helps protect breast cancer cells against oxidative stress by reprogramming their metabolic redox state. Using cell line and patient-derived xenograft models, as well as direct measurements in clinical samples, we have demonstrated the unique regulatory functions of this antisense RNA in increasing the metastatic capacity of breast cancer cells. Results: Non-coding RNAs have emerged as major drivers of metastatic progression. We have recently demonstrated that specific classes of non-coding RNAs, such as tRNAs (Goodarzi et al, Cell, 2016) and tRNA fragments (Goodarzi et al, Cell, 2015), play major roles in breast cancer metastasis as post-transcriptional regulators of gene expression. However, these types of regulatory RNAs constitute only a fraction of the non-coding RNAs that are aberrantly expressed in highly metastatic cells. For example, antisense RNAs are a large but often ignored class of RNAs with poorly understood cellular functions. We recently developed a new computational algorithm to systematically annotate antisense RNAs and identify those that are associated with metastatic progression based on data from cell line and patient-derived xenograft models, as well as matched primary and metastatic tumors from triple-negative breast cancer patients. We identified a previously unknown antisense RNA, which is transcribed from a locus in the 3’ UTR of the gene NQO1 (and is hence named NQO1-AS). Both NQO1-AS and NQO1 are significantly upregulated in highly metastatic breast cancer cells, and we have shown that the NQO1 sense mRNA is stabilized by the expression of NQO1-AS. Our results indicate that NQO1-AS forms a stable duplex with the 3’ UTR of NQO1 and induces the expression of a longer and more stable isoform of NQO1 mRNA. Metabolomic measurements in NQO1 knockdown and control cells revealed that increased NQO1 activity enables cancer cells to better tolerate the oxidative stress experienced during metastasis. We demonstrated this by performing lung metastasis assays in xenograft models. To confirm the clinical relevance of these findings, we performed comprehensive clinical association studies, and also used quantitative PCR and immunohistochemistry to measure NQO1 levels across all disease stages. We observed a highly significant association between higher NQO1 and NQO1-AS expression and metastatic relapse. Methods: We developed a method named iRAs to annotate and quantify antisense RNAs. We used global run-on assays (GRO-seq) and RNA sequencing in poorly and highly metastatic breast cancer cells to identify NQO1-AS as a novel pro-metastatic antisense RNA. We used both Gapmers and CRISPR-i to knock down NQO1-AS and to measure its impact on NQO1 mRNA stability and expression. We used CRISPRi to silence NQO1 in both MDA-231 and HCC-1806 breast cancer lines, and measured the metabolic consequences of NQO1 knockdown by measuring NADPH flux as well as performing general metabolomic profiling. We also used in vivo lung colonization assays (n=5 mice in each arm) to measure the metastatic capacity of NQO1 knockdown cells. Log-rank test (univariate) and Cox Proportional Hazard Models (multivariate) were used to perform survival analyses in METABRIC and the kmplot aggregate dataset. Mann-Whitney or ANOVA was used to compare expression of NQO1 and NQO1-AS across samples stratified based on sub-type and tumor grade/stage in public datasets as well as our own measurements in clinical samples (n=96; 5 healthy, 23 stage I, 30 stage II, 29 stage III, and 9 stage IV). IHC was performed on tissue microarrays from CHTN (Breast Progression), and blinded scoring was used to assess NQO1 levels. Citation Format: Hani Goodarzi, Bruce Culbertson, Kristle Garcia, Lisa Fish. A sense-antisense RNA interaction drives metabolic reprogramming in metastatic 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 PS19-01.

1126 - Genomic analysis of localized prostate cancer identifies AZIN1 as driver of metastatic progression

1126 - Genomic analysis of localized prostate cancer identifies AZIN1 as driver of metastatic progression

Abstract GMM-058: CBX2 IDENTIFIED AS DRIVER OF ANOIKIS ESCAPE AND DISSEMINATION IN HIGH GRADE SEROUS OVARIAN CANCER

Abstract The vast majority of high grade serous ovarian carcinomas (HGSOC) are diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in a variety of cancers. In prostate cancer, CBX2 is a driver of metastatic progression. Little is known about the role of CBX2 in HGSOC. Our hypothesis is that CBX2 upregulation promotes advanced HGSOC by promoting a stem-like transcriptional profile and inhibiting anoikis. Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were queried to establish the role of CBX2 in HGSOC. In vitro evaluation of CBX2 occurred in OVCAR4, PEO1, and OVCAR8 HGSOC cell lines. PolyHEMA-coated plates forced cells to grow in suspension and simulated anoikis-escape. Quantitative polymerase chain reaction and immunoblots evaluated CBX2 expression. Small hairpin RNAs (shRNAs) knocked CBX2 down for loss of function studies. To mimic HGSOC progression several culture conditions, including 2D, colony formation, and 3D, spheroid, were examined. Secreted Gaussia luciferase (gLuc) activity was utilized as an indicator of proliferation. Stemness was tested with the Aldefluor assay, measuring aldehyde dehydrogenase activity (ALDH). Using patient tumors derived from the Gynecology Tissue and Fluid Bank (GTFB) and a HGSOC tissue microarray (TMA) with matched primary, metastatic, and lymph nodes, a CBX2 expression profile was established. Student's t-test was used to define statistical significance, with a p value of < 0.05. Analysis of GEO databases established CBX2 is upregulated in HGSOC tumors compared to benign tissues. Analysis of TCGA found CBX2 expression conveyed worse disease-free survival (11.7 vs 17.6 months, Log-rank test p-value < 0.005) and overall survival (34 vs. 44.8 months, Log-rank test p-value <0.005). Examination of primary HGSOC tumors confirmed CBX2 was upregulated at the protein level in HGSOC compared to benign tissue. In vitro, OVCAR4, PEO1, and OVCAR8 cells upregulate CBX2 when grown in suspension compared to adherent conditions. CBX2 knockdown led to a significant inhibition of proliferation in 2D, 3D, and in suspension. Forced suspension promoted increased ALDH3A1 expression and ALDH activity. CBX2 knockdown led to a decrease in both ALDH3A1 expression and ALDH activity. HGSOC cells grown in suspension were found to be more chemoresistant compared cells grown under adherent conditions. Similarly, knockdown of CBX2 sensitized HGSOC cells to cisplatin. Examination of primary tissue in a TMA of matched patient samples revealed CBX2 is expressed in primary and metastatic disease. We conclude that CBX2 directly impacts proliferation and is overexpressed in HGSOC. Our work indicates that CBX2 is an important regulator of stem-ness, which could play a role in anoikis escape, HGSOC dissemination, and chemoresistance, suggesting that CBX2 may be associated with more advanced disease. This exploration expands our understanding of molecular drivers of HGSOC progression and potentially identifies a novel therapeutic target. Citation Format: Lindsay J. Wheeler, Zachary L. Watson, Lubna Qamar, Alexandra McMellen, Tomomi Yamamoto, Kian Behbakht, and Benjamin G. Bitler. CBX2 IDENTIFIED AS DRIVER OF ANOIKIS ESCAPE AND DISSEMINATION IN HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-058.

P60 - Genomic analysis of localised prostate cancer identifies AZIN1 as driver of metastatic progression

P60 - Genomic analysis of localised prostate cancer identifies AZIN1 as driver of metastatic progression

Abstract PD9-09: Proteomic analysis of extracellular matrix helps define drivers of metastatic progression

Abstract Tumor microenvironment (TME) plays a central role in the development of distant metastasis. However, given its complexity, successful targeting of TME will require a detailed understanding of its composition. Cancer-associated fibroblasts represent a large component of TME and are a major contributor to the extracellular matrix (ECM). Previously, we demonstrated the presence of two fibroblast sub-populations (CDCP1pos vs CD146pos fibroblasts) in breast cancer TME, which determine therapeutic response in estrogen receptor (ER) positive breast cancer. We hypothesized that the same fibroblast subtypes would also influence ECM composition and alter the metastatic potential of breast cancer cells. Here, we present the development of a novel fibroblast driven orthotopic model of ER+ breast cancer metastasis, which we combined with an innovative proteomics approach to precisely quantify extracellular matrix proteins. Results: 1.CDCP1pos fibroblasts promote increased breast cancer cell mobility, invasion and metastasis compared to CD146pos fibroblasts.We demonstrate that CDCP1pos fibroblasts significantly increase the invasion potential of breast cancer cells, when compared to CD146pos fibroblasts. Orthotopic co-implantation of ER+ tumor cells with CDCP1pos fibroblasts into the mammary fat pad of mice more frequently drives distant organ metastases to lung and brain when compared with tumors implanted with CD146pos fibroblasts. 2. Proteomic analysis of ER+ tumors influenced by CDCP1pos fibroblasts revealed known and novel drivers of breast cancer metastasis. Breast cancer cells mixed with CDCP1pos fibroblasts produce a non-uniform collagen orientation to the tumor boarder. Our novel proteomic analysis of TME specific proteins revealed that tumors influenced by CDCP1pos fibroblasts have high expression of many ECM proteins linked to increased risk of breast cancer metastasis, including TNC, FN1, COL5A3, and FBN1 among others. Derived proteomic TME signature accurately predicted lymph node involvement in patients who presented with early stage (T0 and T1) tumors in a cohort of 1,009 breast cancer patients from Cancer Genome Atlas Database. 3. Inhibiting fibroblast production of Tenascin C (TNC) results in decreased breast cancer cell invasion. Our in vitro mixed co-culture models, which contain ER+ breast cancer cells with both fibroblast subtypes, demonstrate that only CDCP1pos fibroblasts produce TNC. Furthermore, in spheroid assays with CDCP1posfibroblasts and breast cancer tumor cells, invasion is inhibited by TNC knockdown. The invasion phenotype can be rescued by addition of EGF, which suggests TNC promotes invasion via EGFR signaling. Conclusion: Metastatic spread of cancer cells relies heavily on TME alterations and makeup of the extracellular matrix. Our data suggest that fibroblast composition directly influences ECM properties and metastatic potential in breast cancer. Taken together, we believe that a better understanding of ECM composition will lead to a more personalized approach to breast cancer treatment. Citation Format: Brechbuhl HM, Barrett AS, Kopin E, Gillen A, Hagen J, Johnson L, Finaly-Shultz J, Sartorius CA, Hansen KC, Kabos P. Proteomic analysis of extracellular matrix helps define drivers of metastatic progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-09.

Abstract B065: Genomic analysis of localized prostate cancer identifies AZIN1 as driver of metastatic progression

Abstract High-risk PCa (HRPCa) remains very heterogeneous with an unacceptable variation in patient outcome after radical prostatectomy, with cancer-specific mortality rates ranging from 4.6% to 20.3% at 10 years’ follow-up. Clearly, we need a better understanding of the tumor biology to enhance the subclassification, enable the identification of lethal PCa, and eventually allow a more precise decision-making regarding treatment. We created a matched case-control study of two clinically identical HRPCa patient groups, both treated with radical prostatectomy but where one developed metastatic recurrence (n=19) and the other did not (n=25), despite very-long-term follow-up. The integrated analysis of copy number aberrations and transcriptome analysis highlighted a focal amplification of 8q22.3 in the metastatic group, associated with a higher expression of antizyme inhibitor 1 (AZIN1) in our cohort. This association of high AZIN1 levels with the metastatic HRPCa phenotype suggests a role for AZIN1 as possible predictor and/or target for metastatic HRPCa. AZIN1 is one of the regulators of the polyamine synthesis, which play a central role in many cellular processes. Our in vitro analyses confirmed that modulation of AZIN1 expression determines both growth and migratory potential of prostate cancer cells. Future experiments are planned to confirm these data in the in vivo setting. RNA sequencing after knockdown of AZIN1 in PCa cells revealed several transcriptional programs that are activated/deactivated upon AZIN1 knockdown. This showed among others a significant upregulation of genes involved in extracellular matrix composition, including genes encoding for subunits of the collagen IV, which is an integral part of the basement membrane. Ongoing experiments focus on identifying the mechanism by which AZIN1 regulates collagen IV expression. Citation Format: Thomas Van den Broeck, Lisa Moris, Thomas Gevaert, Elien Smeets, Stefan Prekovic, Christine Helsen, Hendrik van Poppel, Wouter Everaerts, Christine Buerki, Elai Davicioni, Steven Joniau, Frank Claessens. Genomic analysis of localized prostate cancer identifies AZIN1 as driver of metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B065.

Cell-Cell Contact and Receptor Tyrosine Kinase Signaling.

The behavior of cells within tissues is governed by the activities of adhesion receptors that provide spatial cues and transmit forces through intercellular junctions, and by growth-factor receptors, particularly receptor tyrosine kinases (RTKs), that respond to biochemical signals from the environment. Coordination of these two activities is essential for the patterning and polarized migration of cells during morphogenesis and for homeostasis in mature tissues; loss of this coordination is a hallmark of developing cancer and driver of metastatic progression. Although much is known about the individual functions of adhesion and growth factor receptors, we have a surprisingly superficial understanding of the mechanisms by which their activities are coordinated.

Abstract B49: Epithelial-mesenchymal plasticity primes inherent resistance to targeted therapies

Abstract The metastasis and acquisition of drug resistance in breast cancer cells has been linked to epithelial-mesenchymal transition (EMT). Directed therapies using kinase inhibitors and antibodies to target human epidermal growth factor receptor 2 (Her2) have served as a testament to the potential of molecular-targeted therapies in this breast cancer subtype. However, Her2-targeted therapies are plagued by the pitfalls of intrinsic and acquired resistance, particularly in the metastatic setting. To address these questions we conducted long-term treatments of Her2-transformed cells with TGF-β. This treatment regime resulted in a robust and plastic EMT phenotype as characterized by their CD44/CD24 profile. Following removal of stimuli, cells that had undergone TGF-β-induced EMT enter into a highly heterogeneous state, the importance of which is evidenced by their increased in vivo tumor growth metastatic capability. Importantly, the Lapatinib resistant cell population that arises following TGF-β-induced EMT can be readily eliminated via treatment with recently developed covalent inhibitors of fibroblast growth receptor (FGFR). Overall our data demonstrate that the EMT processes induced by physiologic cytokine stimulation are not only critical drivers of metastatic progression but they also are key events in producing secondary tumors that are poised to be inherently resistant to clinically used targeted molecular therapies. Citation Format: Wells S. Brown, Michael Wendt. Epithelial-mesenchymal plasticity primes inherent resistance to targeted therapies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B49.

The pioneer factor PBX1 is a novel driver of metastatic progression in ERα-positive breast cancer.

Over 30% of ERα breast cancer patients develop relapses and progress to metastatic disease despite treatment with endocrine therapies. The pioneer factor PBX1 translates epigenetic cues and mediates estrogen induced ERα binding. Here we demonstrate that PBX1 plays a central role in regulating the ERα transcriptional response to epidermal growth factor (EGF) signaling. PBX1 regulates a subset of EGF-ERα genes highly expressed in aggressive breast tumours. Retrospective stratification of luminal patients using PBX1 protein levels in primary cancer further demonstrates that elevated PBX1 protein levels correlate with earlier metastatic progression. In agreement, PBX1 protein levels are significantly upregulated during metastatic progression in ERα-positive breast cancer patients. Finally we reveal that PBX1 upregulation in aggressive tumours is partly mediated by genomic amplification of the PBX1 locus. Correspondingly, ERα-positive breast cancer patients carrying PBX1 amplification are characterized by poor survival. Notably, we demonstrate that PBX1 amplification can be identified in tumor derived-circulating free DNA of ERα-positive metastatic patients. Metastatic patients with PBX1 amplification are also characterized by shorter relapse-free survival. Our data identifies PBX1 amplification as a functional hallmark of aggressive ERα-positive breast cancers. Mechanistically, PBX1 amplification impinges on several critical pathways associated with aggressive ERα-positive breast cancer.

A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis.

Cancer metastasis requires that primary tumour cells evolve the capacity to intravasate into the lymphatic system or vasculature, and extravasate into and colonize secondary sites. Others have demonstrated that individual cells within complex populations show heterogeneity in their capacity to form secondary lesions. Here we develop a polyclonal mouse model of breast tumour heterogeneity, and show that distinct clones within a mixed population display specialization, for example, dominating the primary tumour, contributing to metastatic populations, or showing tropism for entering the lymphatic or vasculature systems. We correlate these stable properties to distinct gene expression profiles. Those clones that efficiently enter the vasculature express two secreted proteins, Serpine2 and Slpi, which were necessary and sufficient to program these cells for vascular mimicry. Our data indicate that these proteins not only drive the formation of extravascular networks but also ensure their perfusion by acting as anticoagulants. We propose that vascular mimicry drives the ability of some breast tumour cells to contribute to distant metastases while simultaneously satisfying a critical need of the primary tumour to be fed by the vasculature. Enforced expression of SERPINE2 and SLPI in human breast cancer cell lines also programmed them for vascular mimicry, and SERPINE2 and SLPI were overexpressed preferentially in human patients that had lung-metastatic relapse. Thus, these two secreted proteins, and the phenotype they promote, may be broadly relevant as drivers of metastatic progression in human cancer.

Cell metastasis in Melanoma

Cutaneous melanoma stands out as a model disease on which to study tumor progression and metastasis for several reasons. If undetected or diagnosed late, melanoma is a highly invasive tumor and almost invariably leads to metastatic spread. It tends to metastasize at a time when the tumor burden is low compared to other cancers, which is evident by the size (thickness) of the primary tumor being measured in millimeters. The fact that the majority of the almost 70,000 new melanomas diagnosed in the United States every year are detected when they are still curable, is presumably largely owed to its prominent site of origin, the skin. As a consequence, tissue from early stages of tumor development is relatively easily available for analysis, allowing for the investigation of the whole spectrum of tumor progression and the metastatic process in humans. It is somewhat surprising that comparative genomic approaches to date have not yet consistently identified gene signatures reflecting genes or gene sets that are associated with metastasis or prognosis of melanoma. Nevertheless, tremendous progress has been made in recent years identifying mechanisms leading to metastasis in melanoma. In this review, we highlight some of the key molecules and pathways that have been discovered as drivers of metastatic progression in this disease.