Targeting EMT using low-dose Teniposide by downregulating ZEB2-driven activation of RNA polymerase I in breast cancer.

Metastatic dissemination from the primary tumor is a complex process that requires crosstalk between tumor cells and the surrounding milieu and involves the interplay between numerous cellular-signaling programs. Epithelial-mesenchymal transition (EMT) remains at the forefront of orchestrating a shift in numerous cellular programs, such as stemness, drug resistance, and apoptosis that allow for successful metastasis. Till date, there is limited success in therapeutically targeting EMT. Utilizing a high throughput screen of FDA-approved compounds, we uncovered a novel role of the topoisomerase inhibitor, Teniposide, in reversing EMT. Here, we demonstrate Teniposide as a potent modulator of the EMT program, specifically through an IRF7-NMI mediated response. Furthermore, Teniposide significantly reduces the expression of the key EMT transcriptional regulator, Zinc Finger E-Box Binding Homeobox 2 (ZEB2). ZEB2 downregulation by Teniposide inhibited RNA polymerase I (Pol I) activity and rRNA biogenesis. Importantly, Teniposide treatment markedly reduced pulmonary colonization of breast cancer cells. We have uncovered a novel role of Teniposide, which when used at a very low concentration, mitigates mesenchymal-like invasive phenotype. Overall, its ability to target EMT and rRNA biogenesis makes Teniposide a viable candidate to be repurposed as a therapeutic option to restrict breast cancer metastases.

Ribosome biosynthesis and Hedgehog activity are cooperative actionable signaling mechanisms in breast cancer following radiotherapy.

Hyperactivated ribosome biosynthesis is attributed to a need for elevated protein synthesis that accommodates cell growth and division, and is characterized by nucleomorphometric alterations and increased nucleolar counts. Ribosome biogenesis is challenged when DNA-damaging treatments such as radiotherapy are utilized. Tumor cells that survive radiotherapy form the basis of recurrence, tumor progression, and metastasis. In order to survive and become metabolically revitalized, tumor cells need to reactivate RNA Polymerase I (RNA Pol I) to synthesize ribosomal RNA, an integral component of ribosomes. In this study, we showed that following radiation therapy, tumor cells from breast cancer patients demonstrate activation of a ribosome biosynthesis signature concurrent with enrichment of a signature of Hedgehog (Hh) activity. We hypothesized that GLI1 activates RNA Pol I in response to irradiation and licenses the emergence of a radioresistant tumor population. Our work establishes a novel role for GLI1 in orchestrating RNA Pol I activity in irradiated breast cancer cells. Furthermore, we present evidence that in these irradiated tumor cells, Treacle ribosome biogenesis factor 1 (TCOF1), a nucleolar protein that is important in ribosome biogenesis, facilitates nucleolar translocation of GLI1. Inhibiting Hh activity and RNA Pol I activity disabled the outgrowth of breast cancer cells in the lungs. As such, ribosome biosynthesis and Hh activity present as actionable signaling mechanisms to enhance the effectiveness of radiotherapy.

Hedgehog Signaling Regulates Treg to Th17 Conversion Through Metabolic Rewiring in Breast Cancer.

The tumor immune microenvironment dynamically evolves to support tumor growth and progression. Immunosuppressive regulatory T cells (Treg) promote tumor growth and metastatic seeding in patients with breast cancer. Deregulation of plasticity between Treg and Th17 cells creates an immune regulatory framework that enables tumor progression. Here, we discovered a functional role for Hedgehog (Hh) signaling in promoting Treg differentiation and immunosuppressive activity, and when Hh activity was inhibited, Tregs adopted a Th17-like phenotype complemented by an enhanced inflammatory profile. Mechanistically, Hh signaling promoted O-GlcNAc modifications of critical Treg and Th17 transcription factors, Foxp3 and STAT3, respectively, that orchestrated this transition. Blocking Hh reprogramed Tregs metabolically, dampened their immunosuppressive activity, and supported their transdifferentiation into inflammatory Th17 cells that enhanced the recruitment of cytotoxic CD8+ T cells into tumors. Our results demonstrate a previously unknown role for Hh signaling in the regulation of Treg differentiation and activity and the switch between Tregs and Th17 cells in the tumor microenvironment.

Hedgehog blockade remodels the gut microbiota and the intestinal effector CD8+ T cells in a mouse model of mammary carcinoma.

Given the gut microbiome's rise as a potential frontier in cancer pathogenesis and therapy, leveraging microbial analyses in the study of breast tumor progression and treatment could unveil novel interactions between commensal bacteria and disease outcomes. In breast cancer, the Hedgehog (Hh) signaling pathway is a potential target for treatment due to its aberrant activation leading to poorer prognoses and drug resistance. There are limited studies that have investigated the influences of orally administered cancer therapeutics, such as Vismodegib (a pharmacological, clinically used Hh inhibitor) on the gut microbiota. Using a 4T1 mammary carcinoma mouse model and 16 S rRNA sequencing, we longitudinally mapped alterations in immunomodulating gut microbes during mammary tumor development. Next, we identified changes in the abundance of commensal microbiota in response to Vismodegib treatment of 4T1 mammary tumor-bearing mice. In addition to remodeling gut microbiota, Vismodegib treatment elicited an increase in proliferative CD8+ T cells in the colonic immune network, without any remarkable gastrointestinal-associated side effects. To our knowledge, this is the first study to assess longitudinal changes in the gut microbiome during mammary tumor development and progression. Our study also pioneers an investigation of the dynamic effects of an orally delivered Hh inhibitor on the gut microbiome and the gut-associated immune-regulatory adaptive effector CD8+ T cells. These findings inform future comprehensive studies on the consortium of altered microbes that can impact potential systemic immunomodulatory roles of Vismodegib.

Ribosome Biogenesis: A Central Player in Cancer Metastasis and Therapeutic Resistance.

Ribosomes are a complex ensemble of rRNA and ribosomal proteins that function as mRNA translation machines. Ribosome biogenesis is a multistep process that begins in the nucleolus and concludes in the cytoplasm. The process is tightly controlled by multiple checkpoint and surveillance pathways. Perturbations in these checkpoints and pathways can lead to hyperactivation of ribosome biogenesis. Emerging evidence suggests that cancer cells harbor a specialized class of ribosomes (onco-ribosomes) that facilitates the oncogenic translation program, modulates cellular functions, and promotes metabolic rewiring. Mutations in ribosomal proteins, rRNA processing, and ribosome assembly factors result in ribosomopathies that are associated with an increased risk of developing malignancies. Recent studies have linked mutations in ribosomal proteins and aberrant ribosomes with poor prognosis, highlighting ribosome-targeted therapy as a promising approach for treating patients with cancer. Here, we summarize various aspects of dysregulation of ribosome biogenesis and the impact of resultant onco-ribosomes on malignant tumor behavior, therapeutic resistance, and clinical outcome. Ribosome biogenesis is a promising therapeutic target, and understanding the important determinants of this process will allow for improved and perhaps selective therapeutic strategies to target ribosome biosynthesis.

Quantitative Longitudinal Imaging Reveals that Inhibiting Hedgehog Activity Alleviates the Hypoxic Tumor Landscape.

Metastases account for the majority of mortalities related to breast cancer. The onset and sustained presence of hypoxia strongly correlates with increased incidence of metastasis and unfavorable prognosis in patients with breast cancer. The Hedgehog (Hh) signaling pathway is dysregulated in breast cancer, and its abnormal activity enables tumor progression and metastasis. In addition to programming tumor cell behavior, Hh activity enables tumor cells to craft a metastasis-conducive microenvironment. Hypoxia is a prominent feature of growing tumors that impacts multiple signaling circuits that converge upon malignant progression. We investigated the role of Hh activity in crafting a hypoxic environment of breast cancer. We used radioactive tracer [18F]-fluoromisonidazole (FMISO) positron emission tomography (PET) to image tumor hypoxia. We show that tumors competent for Hh activity are able to establish a hypoxic milieu; pharmacologic inhibition of Hh signaling in a syngeneic mammary tumor model mitigates tumor hypoxia. Furthermore, in hypoxia, Hh activity is robustly activated in tumor cells and institutes increased HIF signaling in a VHL-dependent manner. The findings establish a novel perspective on Hh activity in crafting a hypoxic tumor landscape and molecularly navigating the tumor cells to adapt to hypoxic conditions. IMPLICATIONS: Importantly, we present a translational strategy of utilizing longitudinal hypoxia imaging to measure the efficacy of vismodegib in a preclinical model of triple-negative breast cancer.

Novel role of the dietary flavonoid fisetin in suppressing rRNA biogenesis.

The nucleolus of a cell is a critical cellular compartment that is responsible for ribosome biogenesis and plays a central role in tumor progression. Fisetin, a nutraceutical, is a naturally occurring flavonol from the flavonoid group of polyphenols that has anti-cancer effects. Fisetin negatively impacts several signaling pathways that support tumor progression. However, effect of fisetin on the nucleolus and its functions were unknown. We observed that fisetin is able to physically enter the nucleolus. In the nucleolus, RNA polymerase I (RNA Pol I) mediates the biogenesis of ribosomal RNA. Thus, we investigated the impacts of fisetin on the nucleolus. We observed that breast tumor cells treated with fisetin show a 20-30% decreased nucleolar abundance per cell and a 30-60% downregulation of RNA Pol I transcription activity, as well as a 50-70% reduction in nascent rRNA synthesis, depending on the cell line. Our studies show that fisetin negatively influences MAPK/ERK pathway to impair RNA Pol I activity and rRNA biogenesis. Functionally, we demonstrate that fisetin acts synergistically (CI = 0.4) with RNA Pol I inhibitor, BMH-21 and shows a noteworthy negative impact (60% decrease) on lung colonization of breast cancer cells. Overall, our findings highlight the potential of ribosomal RNA (rRNA) biogenesis as a target for secondary prevention and possible treatment of metastatic disease.

Hedgehog Signaling Regulates Metabolism and Polarization of Mammary Tumor-Associated Macrophages.

Elevated infiltration of immunosuppressive alternatively polarized (M2) macrophages is associated with poor prognosis in patients with cancer. The tumor microenvironment remarkably orchestrates molecular mechanisms that program these macrophages. Here we identify a novel role for oncogenic Hedgehog (Hh) signaling in programming signature metabolic circuitries that regulate alternative polarization of tumor-associated macrophages. Two immunocompetent orthotopic mouse models of mammary tumors were used to test the effect of inhibiting Hh signaling on tumor-associated macrophages. Treatment with the pharmacologic Hh inhibitor vismodegib induced a significant shift in the profile of tumor-infiltrating macrophages. Mass spectrometry-based metabolomic analysis showed Hh inhibition induced significant alterations in metabolic processes, including metabolic sensing, mitochondrial adaptations, and lipid metabolism. In particular, inhibition of Hh in M2 macrophages reduced flux through the UDP-GlcNAc biosynthesis pathway. Consequently, O-GlcNAc-modification of STAT6 decreased, mitigating the immune-suppressive program of M2 macrophages, and the metabolically demanding M2 macrophages shifted their metabolism and bioenergetics from fatty acid oxidation to glycolysis. M2 macrophages enriched from vismodegib-treated mammary tumors showed characteristically decreased O-GlcNAcylation and altered mitochondrial dynamics. These Hh-inhibited macrophages are reminiscent of inflammatory (M1) macrophages, phenotypically characterized by fragmented mitochondria. This is the first report highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. These data describe a novel immunometabolic function of Hh signaling that can be clinically exploited. SIGNIFICANCE: These findings illustrate that Hh activity regulates a metabolic and bioenergetic regulatory program in tumor-associated macrophages that promotes their immune-suppressive polarization.

Disruption of STAT5A and NMI signaling axis leads to ISG20-driven metastatic mammary tumors.

Molecular dynamics of developmental processes are repurposed by cancer cells to support cancer initiation and progression. Disruption of the delicate balance between cellular differentiation and plasticity during mammary development leads to breast cancer initiation and metastatic progression. STAT5A is essential for differentiation of secretory mammary alveolar epithelium. Active STAT5A characterizes breast cancer patients for favorable prognosis. N-Myc and STAT Interactor protein (NMI) was initially discovered as a protein that interacts with various STATs; however, the relevance of these interactions to normal mammary development and cancer was not known. We observe that NMI protein is expressed in the mammary ductal epithelium at the onset of puberty and is induced in pregnancy. NMI protein is decreased in 70% of patient specimens with metastatic breast cancer compared to primary tumors. Here we present our finding that NMI and STAT5A cooperatively mediate normal mammary development. Loss of NMI in vivo caused a decrease in STAT5A activity in normal mammary epithelial as well as breast cancer cells. Analysis of STAT5A mammary specific controlled genetic program in the context of NMI knockout revealed ISG20 (interferon stimulated exonuclease gene 20, a protein involved in rRNA biogenesis) as an unfailing negatively regulated target. Role of ISG20 has never been described in metastatic process of mammary tumors. We observed that overexpression of ISG20 is increased in metastases compared to matched primary breast tumor tissues. Our observations reveal that NMI-STAT5A mediated signaling keeps a check on ISG20 expression via miR-17-92 cluster. We show that uncontrolled ISG20 expression drives tumor progression and metastasis.

Normalizing glucose levels reconfigures the mammary tumor immune and metabolic microenvironment and decreases metastatic seeding.

Obesity and diabetes cumulatively create a distinct systemic metabolic pathophysiological syndrome that predisposes patients to several diseases including breast cancer. Moreover, diabetic and obese women with breast cancer show a significant increase in mortality compared to non-obese and/or non-diabetic women. We hypothesized that these metabolic conditions incite an aggressive tumor phenotype by way of impacting tumor cell-autonomous and tumor cell non-autonomous events. In this study, we established a type 2 diabetic mouse model of triple-negative mammary carcinoma and investigated the effect of a glucose lowering therapy, metformin, on the overall tumor characteristics and immune/metabolic microenvironment. Diabetic mice exhibited larger mammary tumors that had increased adiposity with high levels of O-GlcNAc protein post-translational modification. These tumors also presented with a distinct stromal profile characterized by altered collagen architecture, increased infiltration by tumor-permissive M2 macrophages, and early metastatic seeding compared to non-diabetic/lean mice. Metformin treatment of the diabetic/obese mice effectively normalized glucose levels, reconfigured the mammary tumor milieu, and decreased metastatic seeding. Our results highlight the impact of two metabolic complications of obesity and diabetes on tumor cell attributes and showcase metformin's ability to revert tumor cell and stromal changes induced by an obese and diabetic host environment.

Inhibiting ß-catenin disables nucleolar functions in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) patients with upregulated Wnt/ß-catenin signaling often have poor clinical prognoses. During pathological examinations of breast cancer sections stained for ß-catenin, we made the serendipitous observation that relative to non-TNBC, specimens from TNBC patients have a greater abundance of nucleoli. There was a remarkable direct relationship between nuclear ß-catenin and greater numbers of nucleoli in TNBC tissues. These surprising observations spurred our investigations to decipher the differential functional relevance of the nucleolus in TNBC versus non-TNBC cells. Comparative nucleolar proteomics revealed that the majority of the nucleolar proteins in TNBC cells were potential targets of ß-catenin signaling. Next, we undertook an analysis of the nucleolar proteome in TNBC cells in response to ß-catenin inhibition. This effort revealed that a vital component of pre-rRNA processing, LAS1 like ribosome biogenesis factor (LAS1L) was significantly decreased in the nucleoli of ß-catenin inhibited TNBC cells. Here we demonstrate that LAS1L protein expression is significantly elevated in TNBC patients, and it functionally is important for mammary tumor growth in xenograft models and enables invasive attributes. Our observations highlight a novel function for ß-catenin in orchestrating nucleolar activity in TNBCs.

Isolation of Nucleoli for Characterization of Nucleolar Contents to Uncover Clues to Metastatic Progression.

Nucleolar isolation is a crucial technique for the study of nucleolar contents and regulation of ribosome biogenesis. Lysed cells are spun through various concentrations of sucrose and magnesium chloride to separate the notoriously dense nucleoli from the rest of the cell. Here we describe isolation of nucleoli from the breast cancer cell line MDA-MB-468. The resulting nucleolar fraction is subjected to immunoblotting to confirm the purity of the nucleolar fraction.

Hypoxia re-programs 2'-O-Me modifications on ribosomal RNA.

Hypoxia is one of the critical stressors encountered by various cells of the human body under diverse pathophysiologic conditions including cancer and has profound impacts on several metabolic and physiologic processes. Hypoxia prompts internal ribosome entry site (IRES)-mediated translation of key genes, such as VEGF, that are vital for tumor progression. Here, we describe that hypoxia remarkably upregulates RNA Polymerase I activity. We discovered that in hypoxia, rRNA shows a different methylation pattern compared to normoxia. Heterogeneity in ribosomes due to the diversity of ribosomal RNA and protein composition has been postulated to generate "specialized ribosomes" that differentially regulate translation. We find that in hypoxia, a sub-set of differentially methylated ribosomes recognizes the VEGF-C IRES, suggesting that ribosomal heterogeneity allows for altered ribosomal functions in hypoxia.

Merlin deficiency alters the redox management program in breast cancer.

The expression of Merlin tumor suppressor protein encoded by Neurofibromin 2 (NF2) gene is remarkably decreased in metastatic breast cancer tissues. In order to recapitulate clinical evidence, we generated a unique, conditional Nf2-knockout (Nf2-/- ) mouse mammary tumor model. Merlin-deficient breast tumor cells and Nf2-/- mouse embryonic fibroblasts (MEFs) displayed a robustly invasive phenotype. Moreover, Nf2-/- MEFs presented with notable alterations in redox management networks, implicating a role for Merlin in redox homeostasis. This programmatic alteration resonated with pathways that emerged from breast tumor cells engineered for Merlin deficiency. Further investigations revealed that NF2-silenced cells supported reduced activity of the Nuclear factor, erythroid 2 like 2 antioxidant transcription factor, concomitant with elevated expression of NADPH oxidase enzymes. Importantly, mammary-specific Nf2-/- in an Mouse mammary tumor virus Neu + murine breast cancer model demonstrated accelerated mammary carcinogenesis in vivo. Tumor-derived primary organoids and cell lines were characteristically invasive with evidence of a dysregulated cellular redox management system. As such, Merlin deficiency programmatically influences redox imbalance that orchestrates malignant attributes of mammary/breast cancer.

Hedgehog signaling enables repair of ribosomal DNA double-strand breaks.

Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.

The nucleolus: a central response hub for the stressors that drive cancer progression.

The nucleolus is a sub-nuclear body known primarily for its role in ribosome biogenesis. Increased number and/or size of nucleoli have historically been used by pathologists as a prognostic indicator of cancerous lesions. This increase in nucleolar number and/or size is classically attributed to the increased need for protein synthesis in cancer cells. However, evidences suggest that the nucleolus plays critical roles in many cellular functions in both normal cell biology and disease pathologies, including cancer. As new functions of the nucleolus are elucidated, there is mounting evidence to support the role of the nucleolus in regulating additional cellular functions, particularly response to cellular stressors, maintenance of genome stability, and DNA damage repair, as well as the regulation of gene expression and biogenesis of several ribonucleoproteins. This review highlights the central role of the nucleolus in carcinogenesis and cancer progression and discusses how cancer cells may become "addicted" to nucleolar functions.

Inhibition of Hedgehog signaling reprograms the dysfunctional immune microenvironment in breast cancer.

Host responses to tumor cells include tumor suppressing or promoting mechanisms. We sought to detail the effect of Hedgehog (Hh) pathway inhibition on the composition of the mammary tumor immune portfolio. We hypothesized that Hh signaling mediates a crosstalk between breast cancer cells and macrophages that dictates alternative polarization of macrophages and consequently supports a tumor-promoting microenvironment. We used an immunocompetent, syngeneic mouse mammary cancer model to inhibit Hh signaling with the pharmacological inhibitor, Vismodegib. Using molecular and functional assays, we identified that Hedgehog (Hh) signaling mediates a molecular crosstalk between mammary cancer cells and macrophages that culminates in alternative polarization of macrophages. We carried out an unbiased kinomics and genomics assessment to unravel changes in global kinomic and gene signatures impacted by Hh signaling. Our investigations reveal that in an immunocompetent mammary cancer model, the administration of Vismodegib led to changes in the portfolio of tumor-infiltrating immune cells. This was characterized by a marked reduction in immune-suppressive innate and adaptive cells concomitant with an enrichment of cytotoxic immune cells. Breast cancer cells induce M2 polarization of macrophages via a crosstalk mediated by Hh ligands that alters critical kinomic and genomic signatures. Macrophage depletion improved the benefit of Hedgehog inhibition on eliciting an immunogenic, pro-inflammatory profile. We define a novel role for Hh signaling in disabling anti-tumor immunity. Inhibition of Hh signaling presents with dual advantages of tumor cell-targeting as well as re-educating a dysfunctional tumor microenvironment.

O-GlcNAcylation of GLI transcription factors in hyperglycemic conditions augments Hedgehog activity.

Modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc) promotes tumor cell survival, proliferation, epigenetic changes, angiogenesis, invasion, and metastasis. Here we demonstrate that in conditions of elevated glucose, there is increased expression of key drug resistance proteins (ABCB1, ABCG2, ERCC1, and XRCC1), all of which are regulated by the Hedgehog pathway. In elevated glucose conditions, we determined that the Hedgehog pathway transcription factors, GLI1 and GLI2, are modified by O-GlcNAcylation. This modification functionally enhanced their transcriptional activity. The activity of GLI was enhanced when O-GlcNAcase was inhibited, while inhibiting O-GlcNAc transferase caused a decrease in GLI activity. The metabolic impact of hyperglycemic conditions impinges on maintaining PKM2 in the less active state that facilitates the availability of glycolytic intermediates for biosynthetic pathways. Interestingly, under elevated glucose conditions, PKM2 directly influenced GLI activity. Specifically, abrogating PKM2 expression caused a significant decline in GLI activity and expression of drug resistance proteins. Cumulatively, our results suggest that elevated glucose conditions upregulate chemoresistance through elevated transcriptional activity of the Hedgehog/GLI pathway. Interfering in O-GlcNAcylation of the GLI transcription factors may be a novel target in controlling cancer progression and drug resistance of breast cancer.

Deficiency of tumor suppressor Merlin facilitates metabolic adaptation by co-operative engagement of SMAD-Hippo signaling in breast cancer.

The NF2 gene encodes the tumor and metastasis suppressor protein Merlin. Merlin exerts its tumor suppressive role by inhibiting proliferation and inducing contact-growth inhibition and apoptosis. In the current investigation, we determined that loss of Merlin in breast cancer tissues is concordant with the loss of the inhibitory SMAD, SMAD7, of the TGF-ß pathway. This was reflected as dysregulated activation of TGF-ß signaling that co-operatively engaged with effectors of the Hippo pathway (YAP/TAZ/TEAD). As a consequence, the loss of Merlin in breast cancer resulted in a significant metabolic and bioenergetic adaptation of cells characterized by increased aerobic glycolysis and decreased oxygen consumption. Mechanistically, we determined that the co-operative activity of the Hippo and TGF-ß transcription effectors caused upregulation of the long non-coding RNA Urothelial Cancer-Associated 1 (UCA1) that disengaged Merlin's check on STAT3 activity. The consequent upregulation of Hexokinase 2 (HK2) enabled a metabolic shift towards aerobic glycolysis. In fact, Merlin deficiency engendered cellular dependence on this metabolic adaptation, endorsing a critical role for Merlin in regulating cellular metabolism. This is the first report of Merlin functioning as a molecular restraint on cellular metabolism. Thus, breast cancer patients whose tumors demonstrate concordant loss of Merlin and SMAD7 may benefit from an approach of incorporating STAT3 inhibitors.