Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies.

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.

Necroptosis microenvironment directs lineage commitment in liver cancer.

Primary liver cancer represents a major health problem. It comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which differ markedly with regards to their morphology, metastatic potential and responses to therapy. However, the regulatory molecules and tissue context that commit transformed hepatic cells towards HCC or ICC are largely unknown. Here we show that the hepatic microenvironment epigenetically shapes lineage commitment in mosaic mouse models of liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment determines ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, a function that is conserved in humans. Together, our results provide insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC.

Author Correction: Necroptosis microenvironment directs lineage commitment in liver cancer.

In this Article, the pCaMIN construct consisted of 'mouse MYC and mouse NrasG12V' instead of 'mouse Myc and human NRASG12V; and the pCAMIA construct consisted of 'mouse Myc and human AKT1' instead of 'mouse Myc and Akt1' this has been corrected online.

MYC determines lineage commitment in KRAS-driven primary liver cancer development.

BACKGROUND & AIMS: Primary liver cancer (PLC) comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), two frequent and lethal tumour types that differ regarding their tumour biology and responses to cancer therapies. Liver cells harbour a high degree of cellular plasticity and can give rise to either HCC or iCCA. However, little is known about the cell-intrinsic mechanisms directing an oncogenically transformed liver cell to either HCC or iCCA. The scope of this study was to identify cell-intrinsic factors determining lineage commitment in PLC. METHODS: Cross-species transcriptomic and epigenetic profiling was applied to murine HCCs and iCCAs and to two human PLC cohorts. Integrative data analysis comprised epigenetic Landscape In Silico deletion Analysis (LISA) of transcriptomic data and Hypergeometric Optimization of Motif EnRichment (HOMER) analysis of chromatin accessibility data. Identified candidate genes were subjected to functional genetic testing in non-germline genetically engineered PLC mouse models (shRNAmir knockdown or overexpression of full-length cDNAs). RESULTS: Integrative bioinformatic analyses of transcriptomic and epigenetic data pinpointed the Forkhead-family transcription factors FOXA1 and FOXA2 as MYC-dependent determination factors of the HCC lineage. Conversely, the ETS family transcription factor ETS1 was identified as a determinant of the iCCA lineage, which was found to be suppressed by MYC during HCC development. Strikingly, shRNA-mediated suppression of FOXA1 and FOXA2 with concomitant ETS1 expression fully switched HCC to iCCA development in PLC mouse models. CONCLUSIONS: The herein reported data establish MYC as a key determinant of lineage commitment in PLC and provide a molecular explanation why common liver-damaging risk factors such as alcoholic or non-alcoholic steatohepatitis can lead to either HCC or iCCA. IMPACT AND IMPLICATIONS: Liver cancer is a major health problem and comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), two frequent and lethal tumour types that differ regarding their morphology, tumour biology, and responses to cancer therapies. We identified the transcription factor and oncogenic master regulator MYC as a switch between HCC and iCCA development. When MYC levels are high at the time point when a hepatocyte becomes a tumour cell, an HCC is growing out. Conversely, if MYC levels are low at this time point, the result is the outgrowth of an iCCA. Our study provides a molecular explanation why common liver-damaging risk factors such as alcoholic or non-alcoholic steatohepatitis can lead to either HCC or iCCA. Furthermore, our data harbour potential for the development of better PLC therapies.

microRNA 193a-5p Regulates Levels of Nucleolar- and Spindle-Associated Protein 1 to Suppress Hepatocarcinogenesis.

BACKGROUND & AIMS: We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. METHODS: We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay. RESULTS: Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice. CONCLUSIONS: MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).

Loss of Kmt2c or Kmt2d drives brain metastasis via KDM6A-dependent upregulation of MMP3.

KMT2C and KMT2D, encoding histone H3 lysine 4 methyltransferases, are among the most commonly mutated genes in triple-negative breast cancer (TNBC). However, how these mutations may shape epigenomic and transcriptomic landscapes to promote tumorigenesis is largely unknown. Here we describe that deletion of Kmt2c or Kmt2d in non-metastatic murine models of TNBC drives metastasis, especially to the brain. Global chromatin profiling and chromatin immunoprecipitation followed by sequencing revealed altered H3K4me1, H3K27ac and H3K27me3 chromatin marks in knockout cells and demonstrated enhanced binding of the H3K27me3 lysine demethylase KDM6A, which significantly correlated with gene expression. We identified Mmp3 as being commonly upregulated via epigenetic mechanisms in both knockout models. Consistent with these findings, samples from patients with KMT2C-mutant TNBC have higher MMP3 levels. Downregulation or pharmacological inhibition of KDM6A diminished Mmp3 upregulation induced by the loss of histone-lysine N-methyltransferase 2 (KMT2) and prevented brain metastasis similar to direct downregulation of Mmp3. Taken together, we identified the KDM6A-matrix metalloproteinase 3 axis as a key mediator of KMT2C/D loss-driven metastasis in TNBC.

JAK-STAT Signaling in Inflammatory Breast Cancer Enables Chemotherapy-Resistant Cell States.

Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC. SIGNIFICANCE: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors.

Heterogeneity and transcriptional drivers of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with limited treatment options. To characterize TNBC heterogeneity, we defined transcriptional, epigenetic, and metabolic subtypes and subtype-driving super-enhancers and transcription factors by combining functional and molecular profiling with computational analyses. Single-cell RNA sequencing revealed relative homogeneity of the major transcriptional subtypes (luminal, basal, and mesenchymal) within samples. We found that mesenchymal TNBCs share features with mesenchymal neuroblastoma and rhabdoid tumors and that the PRRX1 transcription factor is a key driver of these tumors. PRRX1 is sufficient for inducing mesenchymal features in basal but not in luminal TNBC cells via reprogramming super-enhancer landscapes, but it is not required for mesenchymal state maintenance or for cellular viability. Our comprehensive, large-scale, multiplatform, multiomics study of both experimental and clinical TNBC is an important resource for the scientific and clinical research communities and opens venues for future investigation.

Untangling the web of intratumour heterogeneity.

Intratumour heterogeneity (ITH) is a hallmark of cancer that drives tumour evolution and disease progression. Technological and computational advances have enabled us to assess ITH at unprecedented depths, yet this accumulating knowledge has not had a substantial clinical impact. This is in part due to a limited understanding of the functional relevance of ITH and the inadequacy of preclinical experimental models to reproduce it. Here, we discuss progress made in these areas and illuminate future directions.

Breast cancer prevention by short-term inhibition of TGFß signaling.

Cancer prevention has a profound impact on cancer-associated mortality and morbidity. We previously identified TGFß signaling as a candidate regulator of mammary epithelial cells associated with breast cancer risk. Here, we show that short-term TGFBR inhibitor (TGFBRi) treatment of peripubertal ACI inbred and Sprague Dawley outbred rats induces lasting changes and prevents estrogen- and carcinogen-induced mammary tumors, respectively. We identify TGFBRi-responsive cell populations by single cell RNA-sequencing, including a unique epithelial subpopulation designated secretory basal cells (SBCs) with progenitor features. We detect SBCs in normal human breast tissues and find them to be associated with breast cancer risk. Interactome analysis identifies SBCs as the most interactive cell population and the main source of insulin-IGF signaling. Accordingly, inhibition of TGFBR and IGF1R decrease proliferation of organoid cultures. Our results reveal a critical role for TGFß in regulating mammary epithelial cells relevant to breast cancer and serve as a proof-of-principle cancer prevention strategy.

The immunological and metabolic landscape in primary and metastatic liver cancer.

The liver is the sixth most common site of primary cancer in humans, and generally arises in a background of cirrhosis and inflammation. Moreover, the liver is frequently colonized by metastases from cancers of other organs (particularly the colon) because of its anatomical location and organization, as well as its unique metabolic and immunosuppressive environment. In this Review, we discuss how the hepatic microenvironment adapts to pathologies characterized by chronic inflammation and metabolic alterations. We illustrate how these immunological or metabolic changes alter immunosurveillance and thus hinder or promote the development of primary liver cancer. In addition, we describe how inflammatory and metabolic niches affect the spreading of cancer metastases into or within the liver. Finally, we review the current therapeutic options in this context and the resulting challenges that must be surmounted.

Platelet PD-L1 reflects collective intratumoral PD-L1 expression and predicts immunotherapy response in non-small cell lung cancer.

Immune-checkpoint inhibitors (ICI) have transformed oncological therapy. Up to 20% of all non-small cell lung cancers (NSCLCs) show durable responses upon treatment with ICI, however, robust markers to predict therapy response are missing. Here we show that blood platelets interact with lung cancer cells and that PD-L1 protein is transferred from tumor cells to platelets in a fibronectin 1, integrin α5ß1 and GPIbα-dependent manner. Platelets from NSCLC patients are found to express PD-L1 and platelet PD-L1 possess the ability to inhibit CD4 and CD8 T-cells. An algorithm is developed to calculate the activation independent adjusted PD-L1 payload of platelets (pPD-L1Adj.), which is found to be superior in predicting the response towards ICI as compared to standard histological PD-L1 quantification on tumor biopsies. Our data suggest that platelet PD-L1 reflects the collective tumor PD-L1 expression, plays important roles in tumor immune evasion and overcomes limitations of histological quantification of often heterogeneous intratumoral PD-L1 expression.

The Worst from Both Worlds: cHCC-ICC.

Prognosis of combined hepatocellular carcinoma-intrahepatic cholangiocarcinoma, a type of primary liver cancer comprising areas with HCC and ICC histopathology, is dismal, and it is unclear if such tumors develop clonally and how they should be treated. In this issue of Cancer Cell, Xue et al. (2019) provide answers to these questions.

PTBP1-Mediated Alternative Splicing Regulates the Inflammatory Secretome and the Pro-tumorigenic Effects of Senescent Cells.

Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer.

Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS.

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.