The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

The survival of patients with cancer has improved significantly, primarily because of multidisciplinary care, improved chemotherapeutic agents in both the adjuvant and metastatic settings, the introduction of targeted biologic agents, and the incorporation of palliative care services into the management scheme. However, despite these advances, a significant proportion of patients continue to experience recurrence after adjuvant treatment, and survival associated with stage IV solid tumors still remains low. A primary or acquired resistance to chemotherapeutic and biologic agents is responsible for the failure of many of the agents used to treat patients with a malignancy. This can be explained by the presence of intratumoral heterogeneity and the molecular complexity of many cancers. Factors contributing to intratumoral heterogeneity include genetic mutations, interactions with the microenvironment-and the presence of cancer stem cells. Cancer stem cells have been identified in a number of solid tumors, including breast cancer, brain tumors, lung cancer, colon cancer, and melanoma. Cancer stem cells have the capacity to self-renew, to give rise to progeny that are different from them, and to utilize common signaling pathways. Cancer stem cells may be the source of all the tumor cells present in a malignant tumor, the reason for the resistance to the chemotherapeutic agent used to treat the malignant tumor, and the source of cells that give rise to distant metastases. This review will focus on properties of cancer stem cells; will compare and contrast the cancer stem cell model with the clonal evolution model of tumorigenesis; will discuss the role of cancer stem cells in the development of resistance to chemotherapy; and will review the therapeutic implications and challenges of targeting cancer stem cells, with an assessment of the potential such an approach holds for improving outcomes for patients with cancer.

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life.

Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.

The standard of care for breast cancer has gradually evolved from empirical treatments based on clinical-pathological characteristics to the use of targeted approaches based on the molecular profile of the tumor. Consequently, an increasing number of molecularly targeted drugs have been developed. These drugs target specific alterations, called driver mutations, which confer a survival advantage to cancer cells. To date, the main challenge remains the identification of predictive biomarkers for the selection of the optimal treatment. On this basis, we evaluated a panel of 25 genes involved in the mechanisms of targeted treatment resistance, in 16 primary breast cancers and their matched recurrences, developed during treatment. Overall, we found a detection rate of mutations higher than that described in the literature. In particular, the most frequently mutated genes were ERBB2 and those involved in the PI3K/AKT/mTOR and the MAPK signaling pathways. The study revealed substantial discordances between primary tumors and metastases, stressing the need for analysis of metastatic tissues at recurrence. We observed that 85.7% of patients with an early-stage or locally advanced primary tumor showed at least one mutation in the primary tumor. This finding could explain the subsequent relapse and might therefore justify more targeted adjuvant treatments. Finally, the mutations detected in 50% of relapsed tissues could have guided subsequent treatment choices in a different way. This study demonstrates that mutation events may be present at diagnosis or arise during cancer treatment. As a result, profiling primary and metastatic tumor tissues may be a major step in defining optimal treatments.

Liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), can be used to understand disease prognosis, tumor heterogeneity, and dynamic response to treatment in metastatic breast cancer (MBC). We explored a novel, 180-gene ctDNA panel and the association of this platform with CTCs and CTC clusters.

KLF4 plays an important role in orchestrating a variety of cellular events, including cell-fate decision, genome stability and apoptosis. Its deregulation is correlated with human diseases such as breast cancer and gastrointestinal cancer. Results from recent biochemical studies have revealed that KLF4 is tightly regulated by posttranslational modifications. Here we report a new finding that KLF4 orchestrates estrogen receptor signaling and facilitates endocrine resistance. We also uncovered the underlying mechanism that alteration of KLF4 by posttranslational modifications such as phosphorylation and ubiquitylation changes tumor cell response to endocrine therapy drugs. IHC analyses using based on human breast cancer specimens showed the accumulation of KLF4 protein in ER-positive breast cancer tissues. Elevated KLF4 expression significantly correlated with prognosis and endocrine resistance. Our drug screening for suppressing KLF4 protein expression led to identification of Src kinase to be a critical player in modulating KLF4-mediated tamoxifen resistance. Depletion of VHL (von Hippel-Lindau tumor suppressor), a ubiquitin E3 ligase for KLF4, reduces tumor cell sensitivity to tamoxifen. We demonstrated phosphorylation of VHL by Src enhances proteolysis of VHL that in turn leads to upregulation of KLF4 and increases endocrine resistance. Suppression of Src-VHL-KLF4 cascade by Src inhibitor or enhancement of VHL-KLF4 ubiquitination by TAT-KLF4 (371-420AAa) peptides re-sensitizes tamoxifen-resistant breast cancer cells to tamoxifen treatment. Taken together, our findings demonstrate a novel role for KLF4 in modulating endocrine resistance via the Src-VHL-KLF4 axis.

Background: Platelets are active players in tumorigenesis, although the exact interactive mechanisms and their direct impact on tumor cells remain largely unknown. Methods: Bidirectional transference of lipids, proteins and RNA between platelets and tumor cells and its impact on tumor cell behavior and tumor process are analyzed in this work. Phenotypic, genetic and functional modifications induced by platelets were analyzed both in tumor cell lines and in circulating tumor cells (CTCs). Results: Data from these assays showed that platelets transferred structural components to tumor cells with higher efficiency than tumor cells to platelets (p = 0.001). This biological interplay occurred by direct contact, internalization or via extracellular vesicles. As a result, tumor cells acquired platelet markers (CD61 and CD42), showed decreased EpCAM, expressed epithelial-to-mesenchymal transition markers, and increased proliferation rates. Moreover, we were able to detect CD61 in CTCs from early and advanced prostate cancer. Conclusions: Our results demonstrated, for the first time, that platelets educate tumor cells by highly efficient transference of lipids, proteins and RNA through different mechanisms. These results suggest that tumor cells and CTCs might acquire highly dynamic and aggressive phenotypes due to platelets interaction including EMT, stem-like phenotype and high proliferative rates.

Constitutively active estrogen receptor α (ER/ESR1) mutations have been identified in approximately one-third of ER+ metastatic breast cancers. Although these mutations are known as mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant but not local recurrences in five independent breast cancer cohorts. In concordance with transcriptomic profiling of ESR1-mutant tumors, genome-edited ESR1 Y537S and D538G-mutant cell models exhibited a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally conferred enhanced cell-cell contacts while decreasing cell-extracellular matrix adhesion. In vivo studies showed ESR1-mutant cells were associated with larger multicellular circulating tumor cell (CTC) clusters with increased compactness compared with ESR1 wild-type CTCs. These preclinical findings translated to clinical observations, where CTC clusters were enriched in patients with ESR1-mutated metastatic breast cancer. Conversely, context-dependent migratory phenotypes revealed cotargeting of Wnt and ER as a vulnerability in a D538G cell model. Mechanistically, mutant ESR1 exhibited noncanonical regulation of several metastatic pathways, including secondary transcriptional regulation and de novo FOXA1-driven chromatin remodeling. Collectively, these data provide evidence for ESR1 mutation-modulated metastasis and suggest future therapeutic strategies for targeting ESR1-mutant breast cancer.

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

Triple negative breast cancers (TNBCs) have a poor prognosis and are not amenable to endocrine- or HER2-targeted therapies. The malignant and invasive feature of TNBCs is correlated with its high cancer stem cell population. Recent results from us and others have unveiled an oncogenic role for the PRMT5-KLF4 axis in regulating tumor progression by orchestrating the stemness in mammary tumor cell as well as genome stability. Methylation of KLF4 by PRMT5 leads to KLF4 stabilization, resulting in promoting mitogenesis.

We describe the genomic landscape of circulating tumour DNA (ctDNA) across pathological subtypes of metastatic breast cancer.

Accumulating evidence demonstrates that circulating tumor cell (CTC) clusters have higher metastatic ability than single CTCs and negatively correlate with cancer patient outcomes. Along with homotypic CTC clusters, heterotypic CTC clusters (such as neutrophil-CTC clusters), which have been identified in both cancer mouse models and cancer patients, lead to more efficient metastasis formation and worse patient outcomes. However, the mechanism by which neutrophils bind to CTCs remains elusive. In this study, we found that intercellular adhesion molecule-1 (ICAM-1) on triple-negative breast cancer (TNBC) cells and CD11b on neutrophils mediate tumor cell-neutrophil binding. Consequently, CD11b deficiency inhibited tumor cell-neutrophil binding and TNBC metastasis. Furthermore, CD11b mediated hydrogen peroxide (H2O2) production from neutrophils. Moreover, we found that ICAM-1 in TNBC cells promotes tumor cells to secrete suPAR, which functions as a chemoattractant for neutrophils. Knockdown of uPAR in ICAM-1+ TNBC cells reduced lung-infiltrating neutrophils and lung metastasis. Bioinformatics analysis confirmed that uPAR is highly expressed in TNBCs, which positively correlates with higher neutrophil infiltration and negatively correlates with breast cancer patient survival. Collectively, our findings provide new insight into how neutrophils bind to CTC to facilitate metastasis and discover a novel potential therapeutic strategy by blocking the ICAM-1-suPAR-CD11b axis to inhibit TNBC metastasis.

Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins. Dynamic hyposialylation in CTCs or deficiency of ST6GAL1 promotes cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in breast cancer. Glycoproteomic analysis reveals newly identified protein substrates of ST6GAL1, such as adhesion or stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As a proof of concept, neutralizing antibodies against one newly identified contributor, PODXL, inhibit CTC cluster formation and lung metastasis associated with paclitaxel treatment for triple-negative breast cancer.

Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model.

A large-panel gene expression analysis was conducted to identify biomarkers associated with the effectiveness of adding palbociclib to fulvestrant.

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.

Inflammatory breast cancer (IBC) is a very aggressive and lethal subtype of breast cancer that accounts for about 4 % of all breast cancers diagnosed in the United States. Despite the efforts of several investigators to identify the molecular factors driving the aggressive phenotype of IBC, a great deal is still unknown about the molecular underpinnings of the disease. In the present study, we investigated the role of interferon-induced transmembrane protein 1 (IFITM1), a well-known interferon-stimulated gene (ISG), in promoting the aggressiveness of SUM149 IBC cells.

Circulating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge.

Traditional factors currently used for prognostic stratification do not always adequately predict treatment response and disease evolution in advanced breast cancer patients. Therefore, the use of blood-based markers, such as circulating tumor cells (CTCs), represents a promising complementary strategy for disease monitoring. In this retrospective study, we explored the role of CTC counts as predictors of disease evolution in breast cancer patients with limited metastatic dissemination.