Combination of tucatinib and neural stem cells secreting anti-HER2 antibody prolongs survival of mice with metastatic brain cancer.

Brain metastases are a leading cause of death in patients with breast cancer. The lack of clinical trials and the presence of the blood-brain barrier limit therapeutic options. Furthermore, overexpression of the human epidermal growth factor receptor 2 (HER2) increases the incidence of breast cancer brain metastases (BCBM). HER2-targeting agents, such as the monoclonal antibodies trastuzumab and pertuzumab, improved outcomes in patients with breast cancer and extracranial metastases. However, continued BCBM progression in breast cancer patients highlighted the need for novel and effective targeted therapies against intracranial metastases. In this study, we engineered the highly migratory and brain tumor tropic human neural stem cells (NSCs) LM008 to continuously secrete high amounts of functional, stable, full-length antibodies against HER2 (anti-HER2Ab) without compromising the stemness of LM008 cells. The secreted anti-HER2Ab impaired tumor cell proliferation in vitro in HER2+ BCBM cells by inhibiting the PI3K-Akt signaling pathway and resulted in a significant benefit when injected in intracranial xenograft models. In addition, dual HER2 blockade using anti-HER2Ab LM008 NSCs and the tyrosine kinase inhibitor tucatinib significantly improved the survival of mice in a clinically relevant model of multiple HER2+ BCBM. These findings provide compelling evidence for the use of HER2Ab-secreting LM008 NSCs in combination with tucatinib as a promising therapeutic regimen for patients with HER2+ BCBM.

Rank ectopic expression in the presence of Neu and MMTV oncogenes alters mammary epithelial cell populations and their tumorigenic potential.

Determination of the mammary epithelial cell that serves as the cell of origin for breast cancer is key to understand tumor heterogeneity and clinical management. In this study, we aimed to decipher whether Rank expression in the presence of PyMT and Neu oncogenes might affect the cell of origin of mammary gland tumors. We observed that Rank expression in PyMT+/- and Neu+/- mammary glands alters the basal and luminal mammary cell populations already in preneoplasic tissue, which may interfere with the tumor cell of origin restricting their tumorigenesis ability upon transplantation assays. In spite of this, Rank expression eventually promotes tumor aggressiveness once tumorigenesis is established.

Repurposing autophagy regulators in brain tumors.

Malignant brain tumors, such as glioblastoma multiforme (GBM) and brain metastases, continue to be an unmet medical challenge. Despite advances in cancer diagnostics and therapeutics, tumor cell colonization in the central nervous system renders most treatment options ineffective. This is primarily due to the selective permeability of the blood-brain barrier (BBB), which hinders the crossing of targeting agents into the brain. As such, repositioning medications that demonstrate anticancer effects and possess the ability to cross the BBB can be a promising option. Antidepressants, which are BBB-permeable, have been reported to exhibit cytotoxicity against tumor cells. Autophagy, specifically, has been identified as one of the common key mediators of antidepressant's antitumor effects. In this work, we provide a comprehensive overview of US Food and Drug Administration (FDA)-approved antidepressants with reported cytotoxic activities in different tumor models, where autophagy dysregulation was demonstrated to play the main part. As such, imipramine, maprotiline, fluoxetine and escitalopram were shown to induce autophagy, whereas nortriptyline, clomipramine and paroxetine were identified as autophagy inhibitors. Sertraline and desipramine, depending on the neoplastic context, were demonstrated to either induce or inhibit autophagy. Collectively, these medications were associated with favorable therapeutic outcomes in a variety of cancer cell models, including brain tumors.

Rankl Impairs Lactogenic Differentiation Through Inhibition of the Prolactin/Stat5 Pathway at Midgestation.

Prolactin and progesterone both orchestrate the proliferation and differentiation of the mammary gland during gestation. Differentiation of milk secreting alveoli depends on the presence of prolactin receptor, the downstream Jak2-Stat5 pathway and the transcription factor Elf5. A strict regulation of Rank signaling is essential for the differentiation of the mammary gland and in particular for alveolar commitment. Impaired alveologenesis and lactation failure are observed in both, knockout and Rank overexpressing mice; however, the underlying molecular mechanism responsible for these phenotypes remains largely unknown. Using genome-wide expression analyses and functional studies, we show here that Rankl (RL) exposure leads to impaired secretory differentiation of alveolar cells not only in MMTV-RANK but also in wild-type (WT) mammary acini. Conversely, pharmacological blockage of Rank signaling at midgestation in WT mice leads to precocious and exacerbated lactogenesis. Mechanistically, RL negatively regulates Stat5 phosphorylation and Elf5 expression at the onset of lactogenesis. Continuous RL exposure leads to the expansion of basal and bipotent cells in WT and MMTV-RANK acini. Overall, we demonstrate that enhanced Rank signaling impairs secretory differentiation during pregnancy by inhibition of the prolactin/p-Stat5 pathway.

APRIL promotes breast tumor growth and metastasis and is associated with aggressive basal breast cancer.

APRIL (a proliferation-inducing ligand) is a cytokine of the tumor necrosis factor family associated mainly with hematologic malignancies. APRIL is also overexpressed in breast carcinoma tissue lesions, although neither its role in breast tumorigenesis nor the underlying molecular mechanism is known. Here, we show that several breast cancer cell lines express APRIL and both its receptors, B cell maturation antigen (BCMA) and transmembrane activator and CAML-interactor (TACI), independently of luminal or basal tumor cell phenotype, and that the mitogen-activated protein kinases p38, ERK1/2, and JNK1/2 are activated in response to APRIL. The inflammatory stimulus poly I:C, a toll-like receptor (TLR) 3 ligand, enhanced APRIL secretion. Silencing experiments decreased cell proliferation, demonstrating that APRIL is a critical autocrine factor for breast tumor growth. Studies of 4T1 orthotopic breast tumors in APRIL transgenic mice showed that an APRIL-enriched environment increased tumor growth and promoted lung metastasis associated with enhanced tumor cell proliferation; BCMA and TACI expression suggests that both participate in these processes. We detected APRIL, BCMA and TACI in human luminal, triple-negative breast carcinomas and HER2 breast carcinomas, with increased levels in more aggressive basal tumors. APRIL was observed near Ki67(+) nuclei and was distributed heterogeneously in the cancer cells, in the leukocyte infiltrate, and in the myoepithelial layer adjacent to the tumor area; these results imply that APRIL provides proliferation signals to tumor cells through paracrine and autocrine signaling. Our study identifies participation of APRIL signaling in breast cancer promotion; we propose impairment of this pathway as a potential therapeutic strategy.

Genetic association study of coronary collateral circulation in patients with coronary artery disease using 22 single nucleotide polymorphisms corresponding to 10 genes involved in postischemic neovascularization.

BACKGROUND: Collateral growth in patients with coronary artery disease (CAD) is highly heterogeneous. Although multiple factors are thought to play a role in collateral development, the contribution of genetic factors to coronary collateral circulation (CCC) is largely unknown. The goal of this study was to assess whether functional single nucleotide polymorphisms (SNPs) in genes involved in vascular growth are associated with CCC. METHODS: 677 consecutive CAD patients were enrolled in the study and their CCC was assessed by the Rentrop method. 22 SNPs corresponding to 10 genes involved in postischemic neovascularization were genotyped and multivariate logistic regression models were adjusted using clinically relevant variables to estimate odds ratios and used to examine associations of allelic variants, genotypes and haplotypes with CCC. RESULTS: Statistical analysis showed that the HIF1A rs11549465 and rs2057482; VEGFA rs2010963, rs1570360, rs699947, rs3025039 and rs833061; KDR rs1870377, rs2305948 and rs2071559; CCL2 rs1024611, rs1024610, rs2857657 and rs2857654; NOS3 rs1799983; ICAM1 rs5498 and rs3093030; TGFB1 rs1800469; CD53 rs6679497; POSTN rs3829365 and rs1028728; and LGALS2 rs7291467 polymorphisms, as well as their haplotype combinations, were not associated with CCC (p < 0.05). CONCLUSIONS: We could not validate in our cohort the association of the NOS3 rs1799983, HIF1A rs11549465, VEGFA rs2010963 and rs699947, and LGALS2 rs7291467 variants with CCC reported by other authors. A validated SNP-based genome-wide association study is required to identify polymorphisms influencing CCC.

Constitutive activation of RANK disrupts mammary cell fate leading to tumorigenesis.

Receptor Activator of NF-kappa B (RANK) pathway controls mammary gland development in mice but its role in mammary stem cell fate remains undefined. We show that constitutive RANK signaling expands luminal and basal mammary compartments including mammary stem and luminal progenitor cell pools and interferes with the generation of CD61+ and Sca1+ luminal cells and Elf5 expression. Impaired mammary cell commitment upon RANK overexpression leads to the accumulation of progenitors including K14+K8+ bipotent cells and the formation of heterogeneous tumors containing hyperplastic basal, luminal, and progenitor cells. RANK expression increases in wild-type mammary epithelia with age and parity, and spontaneous preneoplastic lesions express RANK and accumulate K14+K8+ cells. In human breast tumors, high RANK expression levels are also associated with altered mammary differentiation. These results suggest that increased RANK signaling interferes with mammary cell commitment, contributing to breast carcinogenesis.

Luminal Rank loss decreases cell fitness leading to basal cell bipotency in parous mammary glands.

Rank signaling pathway regulates mammary gland homeostasis and epithelial cell differentiation. Although Rank receptor is expressed by basal cells and luminal progenitors, its role in each individual cell lineage remains unclear. By combining temporal/lineage specific Rank genetic deletion with lineage tracing techniques, we found that loss of luminal Rank reduces the luminal progenitor pool and leads to aberrant alveolar-like differentiation with high protein translation capacity in virgin mammary glands. These Rank-deleted luminal cells are unable to expand during the first pregnancy, leading to lactation failure and impairment of protein synthesis potential in the parous stage. The unfit parous Rank-deleted luminal cells in the alveoli are progressively replaced by Rank-proficient cells early during the second pregnancy, thereby restoring lactation. Transcriptomic analysis and functional assays point to the awakening of basal bipotency after pregnancy by the induction of Rank/NF-κB signaling in basal parous cell to restore lactation and tissue homeostasis.

STING agonist-loaded, CD47/PD-L1-targeting nanoparticles potentiate antitumor immunity and radiotherapy for glioblastoma.

As a key component of the standard of care for glioblastoma, radiotherapy induces several immune resistance mechanisms, such as upregulation of CD47 and PD-L1. Here, leveraging these radiotherapy-elicited processes, we generate a bridging-lipid nanoparticle (B-LNP) that engages tumor-associated myeloid cells (TAMCs) to glioblastoma cells via anti-CD47/PD-L1 dual ligation. We show that the engager B-LNPs block CD47 and PD-L1 and promote TAMC phagocytic activity. To enhance subsequent T cell recruitment and antitumor responses after tumor engulfment, the B-LNP was encapsulated with diABZI, a non-nucleotidyl agonist for stimulator of interferon genes. In vivo treatment with diABZI-loaded B-LNPs induced a transcriptomic and metabolic switch in TAMCs, turning these immunosuppressive cells into antitumor effectors, which induced T cell infiltration and activation in brain tumors. In preclinical murine models, B-LNP/diABZI administration synergized with radiotherapy to promote brain tumor regression and induce immunological memory against glioma. In summary, our study describes a nanotechnology-based approach that hijacks irradiation-triggered immune checkpoint molecules to boost potent and long-lasting antitumor immunity against glioblastoma.

Metixene is an incomplete autophagy inducer in preclinical models of metastatic cancer and brain metastases.

A paucity of chemotherapeutic options for metastatic brain cancer limits patient survival and portends poor clinical outcomes. Using a CNS small-molecule inhibitor library of 320 agents known to be blood-brain barrier permeable and approved by the FDA, we interrogated breast cancer brain metastasis vulnerabilities to identify an effective agent. Metixene, an antiparkinsonian drug, was identified as a top therapeutic agent that was capable of decreasing cellular viability and inducing cell death across different metastatic breast cancer subtypes. This agent significantly reduced mammary tumor size in orthotopic xenograft assays and improved survival in an intracardiac model of multiorgan site metastases. Metixene further extended survival in mice bearing intracranial xenografts and in an intracarotid mouse model of multiple brain metastases. Functional analysis revealed that metixene induced incomplete autophagy through N-Myc downstream regulated 1 (NDRG1) phosphorylation, thereby leading to caspase-mediated apoptosis in both primary and brain-metastatic cells, regardless of cancer subtype or origin. CRISPR/Cas9 KO of NDRG1 led to autophagy completion and reversal of the metixene apoptotic effect. Metixene is a promising therapeutic agent against metastatic brain cancer, with minimal reported side effects in humans, which merits consideration for clinical translation.

The HIF1A C85T single nucleotide polymorphism influences the number of branches of the human coronary tree.

OBJECTIVES: Hypoxia is required for the development of the cardiovascular system. Tissue adaptation to low oxygen is mediated through hypoxia-inducible factor 1. Hypoxia-driven gradients of vascular endothelial growth factor within the heart drive vessel tip sprouting and the angiogenic phase of vasculogenesis. We hypothesized that functional variants of the HIF1A C85T single nucleotide polymorphism (SNP) are associated with the number of coronary artery branches in humans. METHODS: Coronary artery branching in 88 individuals was assessed by dynamic counting of the arterial branches detected in coronary angiograms. Values were classified on the basis of the branches emerging from the right and left coronary arteries. HIF1A C85T genotypes were determined using TaqMan-based assays. A generalized linear model was used to measure the effect of each SNP on the response variables. RESULTS: The presence of the T allele in the HIF1A C85T SNP was associated with few branches of the coronary arteries: 81.03 ± 1.79 for individuals with the CC genotype versus 74.09 ± 2.48 for T-carrying ones (p = 0.042). CONCLUSIONS: The functionality of HIF1A may influence the degree of branching of the human coronary tree. We propose that the HIF1A C85T SNP is a genetic marker that determines interindividual differences in the human coronary artery pattern.

Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma.

PURPOSE: Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. EXPERIMENTAL DESIGN: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. RESULTS: Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. CONCLUSIONS: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

The Network of Cytokines in Brain Metastases.

Brain metastases are the most common of all intracranial tumors and a major cause of death in patients with cancer. Cytokines, including chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors are key regulators in the formation of brain metastases. They regulate the infiltration of different cellular subsets into the tumor microenvironment and affect the therapeutic outcomes in patients. Elucidating the cancer cell-cytokine interactions in the setting of brain metastases is crucial for the development of more accurate diagnostics and efficacious therapies. In this review, we focus on cytokines that are found in the tumor microenvironment of brain metastases and elaborate on their trends of expression, regulation, and roles in cellular recruitment and tumorigenesis. We also explore how cytokines can alter the anti-tumor response in the context of brain metastases and discuss ways through which cytokine networks can be manipulated for diagnosis and treatment.

Polyamines drive myeloid cell survival by buffering intracellular pH to promote immunosuppression in glioblastoma.

Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution.

RANK links senescence to stemness in the mammary epithelia, delaying tumor onset but increasing tumor aggressiveness.

Rank signaling enhances stemness in mouse and human mammary epithelial cells (MECs) and mediates mammary tumor initiation. Mammary tumors initiated by oncogenes or carcinogen exposure display high levels of Rank and Rank pathway inhibitors have emerged as a new strategy for breast cancer prevention and treatment. Here, we show that ectopic Rank expression in the mammary epithelia unexpectedly delays tumor onset and reduces tumor incidence in the oncogene-driven Neu and PyMT models. Mechanistically, we have found that ectopic expression of Rank or exposure to Rankl induces senescence, even in the absence of other oncogenic mutations. Rank leads to DNA damage and senescence through p16/p19. Moreover, RANK-induced senescence is essential for Rank-driven stemness, and although initially translates into delayed tumor growth, eventually promotes tumor progression and metastasis. We uncover a dual role for Rank in the mammary epithelia: Rank induces senescence and stemness, delaying tumor initiation but increasing tumor aggressiveness.

BET inhibition increases ßIII-tubulin expression and sensitizes metastatic breast cancer in the brain to vinorelbine.

Metastases from primary breast cancer result in poor survival. ßIII-tubulin (TUBB3) has been established as a therapeutic target for breast cancer metastases specifically to the brain. In this study, we conducted a systematic analysis to determine the regulation of TUBB3 expression in breast cancer metastases to the brain and strategically target these metastases using vinorelbine (VRB), a drug approved by the U.S. Food and Drug Administration (FDA). We found that human epidermal growth factor receptor 2 (HER2) signaling regulates TUBB3 expression in both trastuzumab-sensitive and trastuzumab-resistant neoplastic cells. We further discovered that bromodomain and extra-terminal domain (BET) inhibition increases TUBB3 expression, rendering neoplastic cells more susceptible to apoptosis by VRB. Orthotopic xenograft assays using two different breast cancer cell models revealed a reduction in tumor volume with BET inhibition and VRB treatment. In addition, in vivo studies using a model of multiple brain metastasis (BM) showed improved survival with the combination of radiation + BET inhibitor (iBET-762) + VRB (75% long-term survivors, P < 0.05). Using in silico analysis and BET inhibition, we found that the transcription factor myeloid zinc finger-1 (MZF-1) protein binds to the TUBB3 promoter. BET inhibition decreases MZF-1 expression and subsequently increases TUBB3 expression. Overexpression of MZF-1 decreases TUBB3 expression and reduces BM in vivo, whereas its knockdown increases TUBB3 expression in breast cancer cells. In summary, this study demonstrates a regulatory mechanism of TUBB3 and provides support for an application of BET inhibition to sensitize breast cancer metastases to VRB-mediated therapy.

Current state of clinical trials in breast cancer brain metastases.

BACKGROUND: Breast cancer brain metastases (BCBM) are the final frontier in neuro-oncology for which more efficacious therapies are required. In this work, we explore clinical trials in BCBM, and determine the shortcomings in the development of new BCBM therapies to shed light on potential areas for enhancement. METHODS: On July 9, 2018, we searched ClinicalTrials.gov for all interventional and therapeutic clinical trials involving BCBM, without limiting for date or location. Information on trial characteristics, including phase, status, start and end dates, study design, primary endpoints, selection criteria, sample size, experimental interventions, results, and publications were collected and analyzed. RESULTS: Fifty-three trials fulfilled the selection criteria. Median trial duration across phases ranged between 3 and 6 years. More than half of the trials were conducted in the United States. Although 94% of the trials were in early phases (I-II), 20% of patients were in phase III trials. Two phase III trials were anteceded by phase II trials that were non-randomized; one reported positive results. Approximately one-third of the trials were completed, whereas 23% of trials were terminated early; mostly due to inadequate enrollment. Only 13% of all trials and 22% of completed trials had published results directly linked to their primary outcomes. CONCLUSIONS: The low number of trials and accrual numbers, the lack of diversity, and the scarcity of published results represent the main troubles in clinical BCBM research. Optimization of BCBM trials is necessary to achieve effective therapies.

FABP7 is a key metabolic regulator in HER2+ breast cancer brain metastasis.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer patients is associated with increased incidence of breast cancer brain metastases (BCBM), but the mechanisms underlying this phenomenon remain unclear. Here, to identify brain-predominant genes critical for the establishment of BCBM, we conducted an in silico screening analysis and identified that increased levels of fatty acid-binding protein 7 (FABP7) correlate with a lower survival and higher incidence of brain metastases in breast cancer patients. We validated these findings using HER2+ BCBM cells compared with parental breast cancer cells. Importantly, through knockdown and overexpression assays, we characterized the role of FABP7 in the BCBM process in vitro and in vivo. Our results uncover a key role of FABP7 in metabolic reprogramming of HER2 + breast cancer cells, supporting a glycolytic phenotype and storage of lipid droplets that enable their adaptation and survival in the brain microenvironment. In addition, FABP7 is shown to be required for upregulation of key metastatic genes and pathways, such as integrins-Src and VEGFA, and for the growth of HER2+ breast cancer cells in the brain microenvironment in vivo. Together, our results support FABP7 as a potential target for the treatment of HER2+ BCBM.

HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma.

The mechanisms by which regulatory T cells (Tregs) migrate to and function within the hypoxic tumor microenvironment are unclear. Our studies indicate that specific ablation of hypoxia-inducible factor 1α (HIF-1α) in Tregs results in enhanced CD8+ T cell suppression versus wild-type Tregs under hypoxia, due to increased pyruvate import into the mitochondria. Importantly, HIF-1α-deficient Tregs are minimally affected by the inhibition of lipid oxidation, a fuel that is critical for Treg metabolism in tumors. Under hypoxia, HIF-1α directs glucose away from mitochondria, leaving Tregs dependent on fatty acids for mitochondrial metabolism within the hypoxic tumor. Indeed, inhibition of lipid oxidation enhances the survival of mice with glioma. Interestingly, HIF-1α-deficient-Treg mice exhibit significantly enhanced animal survival in a murine model of glioma, due to their stymied migratory capacity, explaining their reduced abundance in tumor-bearing mice. Thus HIF-1α acts as a metabolic switch for Tregs between glycolytic-driven migration and oxidative phosphorylation-driven immunosuppression.