ABSTRACT
The metastasis of tumor cells into vital organs is a major cause of death from diverse types of malignancies [...].
ABSTRACT
Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFß and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.
Subject(s)
Breast Neoplasms , Female , Humans , Animals , Mice , Breast Neoplasms/pathology , Signal Transduction , Neoplasm MetastasisABSTRACT
Immune-checkpoint (IC) modulators like the poliovirus receptor (PVR) and programmed death ligand 1 (PD-L1) attenuate innate and adaptive immune responses and are potential therapeutic targets for diverse malignancies, including triple-negative breast cancer (TNBC). The retinoblastoma tumor suppressor, pRB, controls cell growth through E2F1-3 transcription factors, and its inactivation drives metastatic cancer, yet its effect on IC modulators is contentious. Here, we show that RB-loss and high E2F1/E2F2 signatures correlate with expression of PVR, CD274 (PD-L1 gene) and other IC modulators and that pRB represses whereas RB depletion and E2F1 induce PVR and CD274 in TNBC cells. Accordingly, the CDK4/6 inhibitor, palbociclib, suppresses both PVR and PD-L1 expression. Palbociclib also counteracts the effect of CDK4 on SPOP, leading to its depletion, but the overall effect of palbociclib is a net reduction in PD-L1 level. Hydrochloric acid, commonly used to solubilize palbociclib, counteracts its effect and induces PD-L1 expression. Remarkably, lactic acid, a by-product of glycolysis, also induces PD-L1 as well as PVR. Our results suggest a model in which CDK4/6 regulates PD-L1 turnover by promoting its transcription via pRB-E2F1 and degradation via SPOP and that the CDK4/6-pRB-E2F pathway couples cell proliferation with the induction of multiple innate and adaptive immunomodulators, with direct implications for cancer progression, anti-CDK4/6- and IC-therapies.
ABSTRACT
Next-generation sequencing is a superior method for detecting known and novel RNA fusions in formalin-fixed, paraffin-embedded tissue over fluorescence in situ hybridization and RT-PCR. However, confidence in fusion calling and true negatives may be compromised by poor RNA quality. Using a commercial panel of 507 genes and the recommended 3-million read threshold to accept results, two cases yielded false negatives while exceeding this recommendation during clinical validation. To develop a reliable quality control metric that better reflects internal sample quality and improves call confidence, gene expression across 361 patient tumor samples was evaluated to derive a set of 15 genes to serve as a proxy quality control (pQC). These 15 genes were assessed for their normalized expression using the sequencing data from each case and selected for robustness. A threshold of 11 pQC genes produced a 4.71% fail rate, selected for stringency as an acceptable level of repeated testing in the clinical setting, minimizing false-negative calls. To increase the chance that low-quality samples pass pQC, a revision to the library preparation method was also tested, with 75% of previously failed samples passing pQC on resequencing by increasing cDNA input. Taken together, a next-generation sequencing analysis quality control tool is presented that serves as a surrogate for housekeeping genes and improves confidence in fusion calls.
Subject(s)
Molecular Diagnostic Techniques , RNA , Humans , Reproducibility of Results , In Situ Hybridization, Fluorescence , Paraffin Embedding/methods , High-Throughput Nucleotide Sequencing/methodsABSTRACT
BACKGROUND: MDM2 is elevated in multiple myeloma (MM). Although traditionally, MDM2 negatively regulates p53, a growing body of research suggests that MDM2 plays several p53-independent roles in cancer pathogenesis as a regulator of oncogene mRNA stability and translation. Yet, the molecular mechanisms underlying MDM2 overexpression and its role in drug resistance in MM remain undefined. METHODS: Both myeloma cell lines and primary MM samples were employed. Cell viability, cell cycle and apoptosis assays, siRNA transfection, quantitative real-time PCR, immunoblotting, co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), soft agar colony formation and migration assay, pulse-chase assay, UV cross-linking, gel-shift assay, RNA-protein binding assays, MEME-analysis for discovering c-Myc DNA binding motifs studies, reporter gene constructs procedure, gene transfection and reporter assay, MM xenograft mouse model studies, and statistical analysis were applied in this study. RESULTS: We show that MDM2 is associated with poor prognosis. Importantly, its upregulation in primary MM samples and human myeloma cell lines (HMCLs) drives drug resistance. Inhibition of MDM2 by RNAi, or by the MDM2/XIAP dual inhibitor MX69, significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. MDM2 inhibition resulted in a remarkable suppression of relapsed MM cell growth, colony formation, migration and induction of apoptosis through p53-dependent and -independent pathways. Mechanistically, MDM2 was found to reciprocally regulate c-Myc in MM; MDM2 binds to AREs on c-Myc 3'UTR to increase c-Myc mRNA stability and translation, while MDM2 is a direct transcriptional target of c-Myc. MDM2 inhibition rendered c-Myc mRNA unstable, and reduced c-Myc protein expression in MM cells. Importantly, in vivo delivery of MX69 in combination with bortezomib led to significant regression of tumors and prolonged survival in an MM xenograft model. CONCLUSION: Our findings provide a rationale for the therapeutic targeting of MDM2/c-Myc axis to improve clinical outcome of patients with refractory/relapsed MM.
Subject(s)
Leukemia, Myeloid, Acute/pathology , Mutation , Nuclear Proteins/genetics , Proto-Oncogene Proteins c-akt/metabolism , Aged , Female , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Nucleophosmin , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Signal TransductionABSTRACT
The dual phosphatase CDC25 has recently been identified as a target for diverse triple-negative breast cancers including RB1/PTEN/P53-deficient tumors. Moreover, CDC25 inhibitors effectively synergize with PI3K inhibitors to suppress tumor growth. We discuss these findings and the challenges that lie ahead in bringing CDC25 inhibitors to the clinic.
ABSTRACT
CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC), RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Triple Negative Breast Neoplasms/drug therapy , cdc25 Phosphatases/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Inhibitors/therapeutic use , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , cdc25 Phosphatases/genetics , cdc25 Phosphatases/metabolismABSTRACT
A switch from catabolic to anabolic metabolism, a major hallmark of cancer, enables rapid cell duplication, and is driven by multiple oncogenic alterations, including PIK3CA mutation, MYC amplification, and TP53 loss. However, tumor growth requires active mitochondrial function and oxidative phosphorylation (OXPHOS). Recently, loss of the retinoblastoma (RB1) tumor suppressor in breast cancer was shown to induce mitochondrial protein translation (MPT) and OXPHOS. Here, we discuss how increased OXPHOS can enhance anabolic metabolism and cell proliferation, as well as cancer stemness and metastasis. Mitochondrial STAT3, FER/FER-T, and CHCHD2 are also implicated in OXPHOS. We propose that RB1 loss represents a prototypic oncogenic alteration that promotes OXPHOS, that aggressive tumors acquire lethal combinations of oncogenes and tumor suppressors that stimulate anabolism versus OXPHOS, and that targeting both metabolic pathways would be therapeutic.
Subject(s)
Breast Neoplasms/metabolism , Mitochondria/metabolism , Neoplastic Stem Cells/metabolism , Retinoblastoma Binding Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Anabolic Agents/therapeutic use , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Glycolysis/genetics , Humans , Mitochondria/genetics , Neoplasm Metastasis , Neoplastic Stem Cells/pathology , Oxidative Phosphorylation , Retinoblastoma Binding Proteins/deficiency , Ubiquitin-Protein Ligases/deficiencyABSTRACT
Triple-negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which no specific treatment is currently available. Although the retinoblastoma tumor-suppressor gene (RB1) is frequently lost together with TP53 in TNBC, it is not directly targetable. There is thus great interest in identifying vulnerabilities downstream of RB1 that can be therapeutically exploited. Here, we determined that combined inactivation of murine Rb and p53 in diverse mammary epithelial cells induced claudin-low-like TNBC with Met, Birc2/3-Mmp13-Yap1, and Pvt1-Myc amplifications. Gene set enrichment analysis revealed that Rb/p53-deficient tumors showed elevated expression of the mitochondrial protein translation (MPT) gene pathway relative to tumors harboring p53 deletion alone. Accordingly, bioinformatic, functional, and biochemical analyses showed that RB1-E2F complexes bind to MPT gene promoters to regulate transcription and control MPT. Additionally, a screen of US Food and Drug Administration-approved (FDA-approved) drugs identified the MPT antagonist tigecycline (TIG) as a potent inhibitor of Rb/p53-deficient tumor cell proliferation. TIG preferentially suppressed RB1-deficient TNBC cell proliferation, targeted both the bulk and cancer stem cell fraction, and strongly attenuated xenograft growth. It also cooperated with sulfasalazine, an FDA-approved inhibitor of cystine xCT antiporter, in culture and xenograft assays. Our results suggest that RB1 deficiency promotes cancer cell proliferation in part by enhancing mitochondrial function and identify TIG as a clinically approved drug for RB1-deficient TNBC.