Triple-negative breast cancer (TNBC) represents a heterogeneous group of ER- and HER2-negative tumors with poor clinical outcome. We recently reported that Pten-loss cooperates with low expression of ...microRNA-145 to induce aggressive TNBC-like lesions in mice. To systematically identify microRNAs that cooperate with PTEN-loss to induce aggressive human BC, we screened for miRNAs whose expression correlated with PTEN mRNA levels and determined the prognostic power of each PTEN-miRNA pair alone and in combination with other miRs.
Publically available data sets with mRNA, microRNA, genomics, and clinical outcome were interrogated to identify miRs that correlate with PTEN expression and predict poor clinical outcome. Alterations in genomic landscape and signaling pathways were identified in most aggressive TNBC subgroups. Connectivity mapping was used to predict response to therapy.
In TNBC, PTEN loss cooperated with reduced expression of hsa-miR-4324, hsa-miR-125b, hsa-miR-381, hsa-miR-145, and has-miR136, all previously implicated in metastasis, to predict poor prognosis. A subgroup of TNBC patients with PTEN-low and reduced expression of four or five of these miRs exhibited the worst clinical outcome relative to other TNBCs (hazard ratio (HR) = 3.91; P < 0.0001), and this was validated on an independent cohort (HR = 4.42; P = 0.0003). The PTEN-low/miR-low subgroup showed distinct oncogenic alterations as well as TP53 mutation, high RB1-loss signature and high MYC, PI3K, and β-catenin signaling. This lethal subgroup almost completely overlapped with TNBC patients selected on the basis of Pten-low and RB1 signature loss or β-catenin signaling-high. Connectivity mapping predicted response to inhibitors of the PI3K pathway.
This analysis identified microRNAs that define a subclass of highly lethal TNBCs that should be prioritized for aggressive therapy.
FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated ...and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1.
Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index.
Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B.
FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.
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.
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.
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.
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•CDC25 inhibitors target TNBCs that are refractory to CDK4/6 plus CDK2 inhibition•CDC25 mRNA levels are affected by RB1, PTEN, TP53, and MYC status in TNBC•CDC25 plus WEE1 or PI3K inhibitors synergize to effectively suppress growth of TNBCs•Provides a rational for development of CDC25-based therapy for TNBC
Liu et al. report that inhibition of the protein phosphatase CDC25 kills diverse triple-negative breast cancer (TNBC) cells. Moreover, CDC25 antagonists cooperate with other drugs, such as PI3K inhibitors, to efficiently suppress growth of human TNBC engrafted into mice.
Background Cholesterol plays vital roles in human physiology; abnormal levels have deleterious pathological consequences. In cancer, elevated or reduced expression of cholesterol biosynthesis is ...associated with good or poor prognosis, but the underlying mechanisms are largely unknown. The limonoid compounds A1542 and A1543 stimulate ERK/MAPK by direct binding, leading to leukemic cell death and suppression of leukemia in mouse models. In this study, we investigated the downstream consequences of these ERK/MAPK agonists in leukemic cells. Methods We employed RNAseq analysis combined with Q-RT-PCR, western blot and bioinformatics to identify and confirm genes whose expression was altered by A1542 and A1543 in leukemic cells. ShRNA lentiviruses were used to silence gene expression. Cell culture and an animal model (BALB/c) of erythroleukemia induced by Friend virus were utilized to validate effects of cholesterol on leukemia progression. Results RNAseq analysis of A1542-treated cells revealed the induction of all 18 genes implicated in cholesterol biosynthesis. Expression of these cholesterol genes was blocked by cedrelone, an ERK inhibitor. The cholesterol inhibitor lovastatin diminished ERK/MAPK activation by A1542, thereby reducing leukemic cell death induced by this ERK1/2 agonist. Growth inhibition by cholesterol was observed both at the intracellular level, and when orally administrated into a leukemic mouse model. Both HDL and LDL also suppressed leukemogenesis, implicating these lipids as important prognostic markers for leukemia progression. Mechanistically, knockdown experiments revealed that the activation of SREBP1/2 by A1542-A1543 was responsible for induction of only a sub-set of cholesterol biosynthesis genes. Induction of other regulatory factors by A1542-A1543 including EGR1, AP1 (FOS + JUN) LDLR, IER2 and others may cooperate with SREBP1/2 to induce cholesterol genes. Indeed, pharmacological inhibition of AP1 significantly inhibited cholesterol gene expression induced by A1542. In addition to leukemia, high expression of cholesterol biosynthesis genes was found to correlate with better prognosis in renal cancer. Conclusions This study demonstrates that ERK1/2 agonists suppress leukemia and possibly other types of cancer through transcriptional stimulation of cholesterol biosynthesis genes. Keywords: Drug screening, ERK1/2 agonist compounds, Apoptosis, Leukemia inhibition, Cholesterol biosynthesis, SREBP1/2, AP1
Wiskott-Aldrich Syndrome, WAS/WAVE, is a rare, X-linked immune-deficiency disease caused by mutations in the
gene, which together with its homolog, N-
, regulates actin cytoskeleton remodeling and ...cell motility. WAS patients suffer from microthrombocytopenia, characterized by a diminished number and size of platelets, though the underlying mechanism is not fully understood. Here, we identified FLI1 as a direct transcriptional regulator of
and its binding partner
. Depletion of either
or
in human erythroleukemic cells accelerated cell proliferation, suggesting tumor suppressor function of both genes in leukemia. Depletion of
also led to a significant reduction in the percentage of CD41 and CD61 positive cells, which mark committed megakaryocytes. RNAseq analysis revealed common changes in megakaryocytic gene expression following FLI1 or WASP knockdown. However, in contrast to FLI1, WASP depletion did not alter expression of late-stage platelet-inducing genes. N-WASP was not regulated by FLI1, yet its silencing also reduced the percentage of CD41+ and CD61+ megakaryocytes. Moreover, combined knockdown of WASP and N-WASP further suppressed megakaryocyte differentiation, indicating a major cooperation of these related genes in controlling megakaryocytic cell fate. However, unlike WASP/WIP, N-WASP loss suppressed leukemic cell proliferation. WASP, WIP and N-WASP depletion led to induction of FLI1 expression, mediated by GATA1, and this may mitigate the severity of platelet deficiency in WAS patients. Together, these results uncover a crucial role for FLI1 in megakaryocyte differentiation, implicating this transcription factor in regulating microthrombocytopenia associated with Wiskott-Aldrich syndrome.
Breast cancer is a highly heterogeneous disease, with several different subtypes being characterized by distinct histology, gene expression patterns, and genetic alterations. The tumor suppressor ...gene retinoblastoma 1 (RB1) is frequently lost in both luminal-B and triple-negative tumor (TNT; i.e., estrogen receptor-, progesterone receptor-, and human epidermal growth factor receptor 2-negative) breast cancer subtypes. However, a causal role for RB1 loss in different subtypes remains undefined. Here we report that deletion of Rb alone or together with its relative p107 in mouse mammary stem/bipotent progenitor cells induced focal acinar hyperplasia with squamous metaplasia. These lesions progressed into histologically diverse, transplantable mammary tumors with features of either luminal-B or TNT subtypes. The TNTs included basal-like tumors as well as tumors that exhibited epithelial-to-mesenchymal transition (EMT). The EMT-type tumors and a subset of the basal-like tumors, but not luminal-B-like tumors, expressed mutant forms of the tumor suppressor p53. Accordingly, targeted deletion of both Rb and p53 in stem/bipotent progenitors led to histologically uniform, aggressive, EMT-type tumors. Reintroduction of Rb into these tumor cells suppressed growth in vitro and tumor formation in vivo. These results establish a causal role for Rb loss in breast cancer in mice and demonstrate that cooperating oncogenic events, such as mutations in p53, dictate tumor subtype after Rb inactivation.
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, TGFβ 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.
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