Induction of Aryl Hydrocarbon Receptor-Mediated Cancer Cell-Selective Apoptosis in Triple-Negative Breast Cancer Cells by a High-Affinity Benzimidazoisoquinoline.

Triple-negative breast cancer (TNBC) remains a disease with a paucity of targeted treatment opportunities. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in a wide range of physiological processes, including the sensing of xenobiotics, immune function, development, and differentiation. Different small-molecule AhR ligands drive strikingly varied cellular and organismal responses. In certain cancers, AhR activation by select small molecules induces cell cycle arrest or apoptosis via activation of tumor-suppressive transcriptional programs. AhR is expressed in triple-negative breast cancers, presenting a tractable therapeutic opportunity. Here, we identify a novel ligand of the aryl hydrocarbon receptor that potently and selectively induces cell death in triple-negative breast cancer cells and TNBC stem cells via the AhR. Importantly, we found that this compound, Analog 523, exhibits minimal cytotoxicity against multiple normal human primary cells. Analog 523 represents a high-affinity AhR ligand with potential for future clinical translation as an anticancer agent.

Oxidized analogs of Di(1H-indol-3-yl)methyl-4-substituted benzenes are NR4A1-dependent UPR inducers with potent and safe anti-cancer activity.

Di(1H-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF3) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF3, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X+ OMs-s) having CF3, CO2Me and Cl groups were more effective inhibitors of cancer cell viability than their precursors. 19F NMR spectroscopy and differential scanning calorimetry strongly indicated interactions of DIM-Ph-4-CF3+ OMs- with the NR4A1 ligand binding domain, and compound-induced apoptosis of prostate cancer cells was dependent on NR4A1. DIM-Ph-4-CF3+ OMs- showed robust inhibition of LNCaP prostate cancer xenografts with no apparent toxicity. In vitro and in vivo, DIM-Ph-4-CF3+ OMs- activated proapoptotic unfolded protein response (UPR) signaling in prostate cancer cells. Independently of DIM-Ph-4-CF3+ OMs-, the bulk of NR4A1 localized to the cytoplasm in various cancer cell lines, suggesting a cytoplasmic mechanism-of-action of DIM-Ph-4-CF3+ OMs- in UPR induction and cell death. In summary, the data suggest that oxidized analogs of DIM-Ph-4-CF3 possess potent and safe anti-cancer activity which is mediated through UPR signaling downstream of NR4A1 binding.

ERN1 and ALPK1 inhibit differentiation of bi-potential tumor-initiating cells in human breast cancer.

Cancers are heterogeneous by nature. While traditional oncology screens commonly use a single endpoint of cell viability, altering the phenotype of tumor-initiating cells may reveal alternative targets that regulate cellular growth by processes other than apoptosis or cell division. We evaluated the impact of knocking down expression of 420 kinases in bi-lineage triple-negative breast cancer (TNBC) cells that express characteristics of both myoepithelial and luminal cells. Knockdown of ERN1 or ALPK1 induces bi-lineage MDA-MB-468 cells to lose the myoepithelial marker keratin 5 but not the luminal markers keratin 8 and GATA3. In addition, these cells exhibit increased ß-casein production. These changes are associated with decreased proliferation and clonogenicity in spheroid cultures and anchorage-independent growth assays. Confirmation of these assays was completed in vivo, where ERN1- or ALPK1-deficient TNBC cells are less tumorigenic. Finally, treatment with K252a, a kinase inhibitor active on ERN1, similarly impairs anchorage-independent growth of multiple breast cancer cell lines. This study supports the strategy to identify new molecular targets for types of cancer driven by cells that retain some capacity for normal differentiation to a non-tumorigenic phenotype. ERN1 and ALPK1 are potential targets for therapeutic development.

Design and Characterization of Novel EphA2 Agonists for Targeted Delivery of Chemotherapy to Cancer Cells.

The development of novel, targeted delivery agents for anti-cancer therapies requires the design and optimization of potent and selective tumor-targeting agents that are stable and amenable to conjugation with chemotherapeutic drugs. While short peptides represent potentially an excellent platform for these purposes, they often get degraded and are eliminated too rapidly in vivo. In this study, we used a combination of nuclear magnetic resonance-guided structure-activity relationships along with biochemical and cellular studies to derive a novel tumor-homing agent, named 123B9, targeting the EphA2 tyrosine kinase receptor ligand-binding domain. Conjugating 123B9 to the chemotherapeutic drug paclitaxel (PTX) via a stable linker results in an agent that is significantly more effective than the unconjugated drug in both a pancreatic cancer xenograft model and a melanoma lung colonization and metastases model. Hence, 123B9 could represent a promising strategy for the development of novel targeted therapies for cancer.

Transcription factor Ets1, but not the closely related factor Ets2, inhibits antibody-secreting cell differentiation.

B cell differentiation into antibody-secreting cells (ASCs) is a tightly regulated process under the control of multiple transcription factors. One such transcription factor, Ets1, blocks the transition of B cells to ASCs via two separate activities: (i) stimulating the expression of target genes that promote B cell identity and (ii) interfering with the functional activity of the transcription factor Blimp1. Ets1 is a member of a multigene family, several members of which are expressed within the B cell lineage, including the closely related protein Ets2. In this report, we demonstrate that Ets1, but not Ets2, can block ASC formation despite the fact that Ets1 and Ets2 bind to apparently identical DNA sequence motifs and are thought to regulate overlapping sets of target genes. The DNA binding domain of Ets1 is required, but not sufficient by itself, to block ASC formation. In addition, less conserved regions within the N terminus of Ets1 play an important role in inhibiting B cell differentiation. Differences between the N termini of Ets1 and Ets2, rather than differences in the DNA binding domains, determine whether the proteins are capable of blocking ASC formation or not.

ROCK1 inhibition promotes the self-renewal of a novel mouse mammary cancer stem cell.

The differentiation of stem-like tumor cells may contribute to the cellular heterogeneity of breast cancers. We report the propagation of highly enriched mouse mammary cancer stem cells that retain the potential to differentiate both in vivo and in culture and their use to identify chemical compounds that influence both self-renewal and differentiation. We identify epithelial tumor-initiating cells (ETICs) that express lineage markers of both basal and luminal mammary cell lineages and retain the potential, from even single cells, to generate heterogeneous tumors similar to the tumor of origin. ETICs can progress through a Rho-associated coiled-coil containing protein kinase 1 dependent, epithelial to mesenchymal transition to generate mesenchymal tumor-initiating cells capable of initiating tumors of limited heterogeneity. The propagation of ETICs may allow for the identification of new therapeutic compounds that may inhibit or prevent progression of some types of breast cancer.

Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors.

Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR(+). However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca(2+) transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis.

Analogues of orphan nuclear receptor small heterodimer partner ligand and apoptosis inducer (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid. 2. Impact of 3-chloro group replacement on inhibition of proliferation and induction of apoptosis of leukemia and cancer cell lines.

The parent phenol of adapalene and its (E)-cinnamic acid analogue were found to induce cancer cell apoptosis but cause adverse systemic effects when administered to mice. In contrast, their respective 5-Cl- and 3-Cl-substituted analogues had their adverse effects mitigated without a comparable loss of cancer cell inhibitory activity. As a result, pharmacologic space in this region of the cinnamic phenyl ring scaffold was explored. Various substituents were introduced, and their effects on cancer cell proliferation and viability were evaluated. Cinnamic acids having 3-Br, CN, NO(2), NH(2), OMe, and N(3) groups had activity comparable to that of 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid. A comparative molecular field analysis study indicated that introduction of an H-bond acceptor at position 3 of the central phenyl ring would favor inhibition of leukemia cell viability, and docking suggested its hydrogen bonding with a polar group in a small heterodimer partner homology model. The 3-CN, NO(2), NH(2), and OH analogues also inhibited MMTV-Wnt1 murine mammary stem cell viability.

Heteroatom-substituted analogues of orphan nuclear receptor small heterodimer partner ligand and apoptosis inducer (E)-4-[3-(1-Adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid.

(E)-4-[3'-(1-Adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces the cell cycle arrest and apoptosis of cancer cells. Because its pharmacologic properties-solubility, bioavailability, and toxicity-required improvement for translation, structural modifications were made by introducing nitrogen atoms into the cinnamyl ring and replacing its E-double bond with XCH(2) (X = O, N, and S) with the objective of enhancing these properties without impacting apoptosis-inducing activity. Analogues having nitrogen atoms in heterocyclic rings corresponding to the cinnamyl phenyl ring displayed equal or higher biological activities. The pyrimidine and pyridine analogues were more soluble in both phosphate-buffered saline and water. While the 2,5-disubstituted pyridine analogue was the most potent inducer of KG-1 acute myeloid leukemia cell apoptosis, on the basis of apoptotic activity in KG-1 cells and solubility, the 2,5-disubstituted pyrimidine proved to be the more promising candidate for treatment of acute myeloid leukemia.

Ets2 regulates colonic stem cells and sensitivity to tumorigenesis.

Ets2 has both tumor repressive and supportive functions for different types of cancer. We have investigated the role of Ets2 within intestinal epithelial cells in postnatal mouse colon development and tumorigenesis. Conditional inactivation of Ets2 within intestinal epithelial cells results in over representation of Ets2-deficient colon crypts within young and adult animals. This preferential representation is associated with an increased number of proliferative cells within the stem cell region and an increased rate of crypt fission in young mice that result in larger patches of Ets2-deficient crypts. These effects are consistent with a selective advantage of Ets2-deficient intestinal stem cells in colonizing colonic crypts and driving crypt fission. Ets2-deficient colon crypts have an increased mucosal thickness, an increased number of goblet cells, and an increased density. Mice with Ets2-deficient intestinal cells develop more colon tumors in response to treatment with azoxymethane and dextran sulfate sodium. The selective population of colon crypts, the altered differentiation state and increased sensitivity to carcinogen-induced tumors all indicate that Ets2 deficiency alters colon stem cell number or behavior. Ets2-dependent, epithelial cell-autonomous repression of intestinal tumors may contribute to protection from colon cancer of persons with increased dosage of chromosome 21.

Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo.

Maternal embryonic leucine zipper kinase (MELK) is expressed in several developing tissues, in the adult germ line, and in adult neural progenitors. MELK expression is elevated in aggressive undifferentiated tumors, correlating with poor patient outcome in human breast cancer. To investigate the role of MELK in mammary tumorigenesis in vivo, we used a MELK-green fluorescent protein (GFP) reporter mouse, which allows prospective isolation of MELK-expressing cells based on GFP fluorescence. We found that in the normal mammary gland, cells expressing high levels of MELK were enriched in proliferating cells that express markers of mammary progenitors. The isolation of cells with high levels of MELK in mammary tumors from MMTV-Wnt1/MELK-GFP bitransgenic mice resulted in a significant enrichment of tumorsphere formation in culture and tumor initiation after transplantation into mammary fat pads of syngeneic mice. Furthermore, using lentiviral delivery of MELK-specific shRNA and limiting dilution cell transplantations, we showed that MELK function is required for mammary tumorigenesis in vivo. Our findings identify MELK as a potential target in breast tumor-initiating cells.

Postnatal tissue-specific disruption of transcription factor FoxN1 triggers acute thymic atrophy.

The transcription factor FoxN1 is essential for differentiation of thymic epithelial cell (TEC) progenitors during thymic organogenesis. However, limited information is available on the postnatal contribution of FoxN1 to thymic maintenance. To address this question, we generated a loxP-floxed FoxN1 (fx) mouse with three different promoter-driven inducible CreER(T) transgenes. Postnatal ubiquitous deletion of FoxN1 caused dramatic thymic atrophy in 5 days and more severe deterioration in medullary TECs (mTECs) than in cortical TECs (cTECs). Induction of FoxN1 deletion selectively in K5 promoter-driven somatic epithelial cells (mostly mTECs and possibly some adult epithelial stem cells) was sufficient to cause significant thymic atrophy, whereas FoxN1 deletion in K18 promoter-driven somatic epithelial cells (mostly cTECs) was not. Thymic atrophy resulted from increased apoptosis and was associated with activation of the p53 gene in mature mTECs. Although FoxN1 is required for the development of both mTECs and cTECs in thymic organogenesis, it is most important for the maintenance of mTECs in the postnatal thymus, which are in turn necessary to prevent thymic atrophy.

Placenta-specific gene activation and inactivation using integrase-defective lentiviral vectors with the Cre/LoxP system.

Transgenic and knockout studies have advanced our understanding of the genetic control of embryonic development over the past decades. However, interpretation of the phenotype of mutant mice is potentially complicated, since the commonly used knockout approach modifies both the fetal and placental genome. To circumvent this problem, we previously developed a placenta-specific gene manipulation system by lentiviral vector transduction of embryos at the blastocyst stage. In the present study, by combination with the Cre/LoxP system, we successfully demonstrate placenta-specific gene activation and inactivation in EGFP reporter mice and Ets2 floxed mice, respectively. Transient expression using integrase-defective lentiviral (IDLV) vectors diminished the toxic effect of Cre expression and solved the dilemma of mosaic recombination with lower concentrations and toxic effects with higher concentrations of Cre recombinase. We also show that placenta-specific Ets2 disruption causes embryonic lethality and reconfirmed the critical role of Ets2 during placentation. This technology facilitates both gain and loss of gene function analyses in placental development during pregnancy. Since IDLV vectors can efficiently transduce a variety of cell types similarly to wild-type vectors, our IDLV-Cre strategy is potentially useful for a wide range of applications.

Ets1 and Ets2 are required for endothelial cell survival during embryonic angiogenesis.

The ras/Raf/Mek/Erk pathway plays a central role in coordinating endothelial cell activities during angiogenesis. Transcription factors Ets1 and Ets2 are targets of ras/Erk signaling pathways that have been implicated in endothelial cell function in vitro, but their precise role in vascular formation and function in vivo remains ill-defined. In this work, mutation of both Ets1 and Ets2 resulted in embryonic lethality at midgestation, with striking defects in vascular branching having been observed. The action of these factors was endothelial cell autonomous as demonstrated using Cre/loxP technology. Analysis of Ets1/Ets2 target genes in isolated embryonic endothelial cells demonstrated down-regulation of Mmp9, Bcl-X(L), and cIAP2 in double mutants versus controls, and chromatin immunoprecipitation revealed that both Ets1 and Ets2 were loaded at target promoters. Consistent with these observations, endothelial cell apoptosis was significantly increased both in vivo and in vitro when both Ets1 and Ets2 were mutated. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis.

Cell cycle-dependent variation of a CD133 epitope in human embryonic stem cell, colon cancer, and melanoma cell lines.

CD133 (Prominin1) is a pentaspan transmembrane glycoprotein expressed in several stem cell populations and cancers. Reactivity with an antibody (AC133) to a glycoslyated form of CD133 has been widely used for the enrichment of cells with tumor-initiating activity in xenograph transplantation assays. We have found by fluorescence-activated cell sorting that increased AC133 reactivity in human embryonic stem cells, colon cancer, and melanoma cells is correlated with increased DNA content and, reciprocally, that the least reactive cells are in the G(1)-G(0) portion of the cell cycle. Continued cultivation of cells sorted on the basis of high and low AC133 reactivity results in a normalization of the cell reactivity profiles, indicating that cells with low AC133 reactivity can generate highly reactive cells as they resume proliferation. The association of AC133 with actively cycling cells may contribute to the basis for enrichment for tumor-initiating activity.

Contrasting expression of keratins in mouse and human embryonic stem cells.

RNA expression data reveals that human embryonic stem (hES) cells differ from mouse ES (mES) cells in the expression of RNAs for keratin intermediate filament proteins. These differences were confirmed at the cellular and protein level and may reflect a fundamental difference in the epithelial nature of embryonic stem cells derived from mouse and human blastocysts. Mouse ES cells express very low levels of the simple epithelial keratins K8, K18 and K19. By contrast hES cells express moderate levels of the RNAs for these intermediate filament proteins as do mouse stem cells derived from the mouse epiblast. Expression of K8 and K18 RNAs are correlated with increased c-Jun RNA expression in both mouse and human ES cell cultures. However, decreasing K8 and K18 expression associated with differentiation to neuronal progenitor cells is correlated with increasing expression of the Snai2 (Slug) transcriptional repression and not decreased Jun expression. Increasing K7 expression is correlated with increased CDX2 and decreased Oct4 RNA expression associated with the formation of trophoblast derivatives by hES cells. Our study supports the view that hES cells are more similar to mouse epiblast cells than mouse ES cells and is consistent with the epithelial nature of hES cells. Keratin intermediate filament expression in hES cells may modulate sensitivity to death receptor mediated apoptosis and stress.

T-cadherin supports angiogenesis and adiponectin association with the vasculature in a mouse mammary tumor model.

T-cadherin delineates endothelial, myoepithelial, and ductal epithelial cells in the normal mouse mammary gland, and becomes progressively restricted to the vasculature during mammary tumorigenesis. To test the function of T-cadherin in breast cancer, we inactivated the T-cadherin (Cdh13) gene in mice and evaluated tumor development and pathology after crossing the mutation into the mouse mammary tumor virus (MMTV)-polyoma virus middle T (PyV-mT) transgenic model. We report that T-cadherin deficiency limits mammary tumor vascularization and reduces tumor growth. Tumor transplantation experiments confirm the stromal role of T-cadherin in tumorigenesis. In comparison with wild-type MMTV-PyV-mT controls, T-cadherin-deficient tumors are pathologically advanced and metastasize to the lungs. T-cadherin is a suggested binding partner for high molecular weight forms of the circulating, fat-secreted hormone adiponectin. We discern adiponectin in association with the T-cadherin-positive vasculature in the normal and malignant mammary glands and report that this interaction is lost in the T-cadherin null condition. This work establishes a role for T-cadherin in promoting tumor angiogenesis and raises the possibility that vascular T-cadherin-adiponectin association may contribute to the molecular cross-talk between tumor cells and the stromal compartment in breast cancer.

Keratin overexpression levels correlate with the extent of spontaneous pancreatic injury.

Mutation of the adult hepatocyte keratins K8 and K18 predisposes to liver disease. In contrast, exocrine pancreas K8 and K18 are dispensable and are co-expressed with limited levels of membrane-proximal K19 and K20. Overexpression of mutant K18 or genetic ablation of K8 in mouse pancreas is well tolerated whereas overexpression of K8 causes spontaneous chronic pancreatitis. To better understand the effect of exocrine pancreatic keratin overexpression, we compared transgenic mice that overexpress K18, K8, or K8/K18, associated with minimal, modest, or large increases in keratin expression, respectively, with nontransgenic wild-type (WT) mice. Overexpression of the type-II keratin K8 up-regulated type-I keratins K18, K19, and K20 and generated K19/K20-containing neocytoplasmic typical or short filaments; however, overexpression of K18 had no effect on K8 levels. K8- and K18-overexpressing pancreata were histologically similar to WT, whereas K8/K18 pancreata displayed age-enhanced vacuolization and atrophy of the exocrine pancreas and exhibited keratin hyperphosphorylation. Zymogen granules in K8/K18 pancreata were 50% smaller and more dispersed than their normal apical concentration but were twice as numerous as in WT controls. Therefore, modest keratin overexpression has minor effects on the exocrine pancreas whereas significant keratin overexpression alters zymogen granule organization and causes aging-associated exocrine atrophy. Keratin absence or mutation is well tolerated after pancreatic but not liver injury, whereas excessive overexpression is toxic to the pancreas but not the liver when induced under basal conditions.

Anoikis effector Bit1 negatively regulates Erk activity.

Bcl-2 inhibitor of transcription (Bit1) is a mitochondrial protein that functions as a peptidyl-tRNA hydrolase, but, when released into the cytoplasm, it elicits apoptosis. The proapoptotic function is uniquely counteracted by integrin-mediated cell attachment. We generated a conditional KO mouse of the Bit1 gene by using the Cre-LoxP recombination system. Bit1-null mice were born alive but with some developmental abnormalities. They developed a runting syndrome after birth and died within the first 2 weeks. Cultured fibroblasts from the Bit1-null embryos [mouse embryo fibroblasts (MEFs)] were more resistant to cell death induced by loss of attachment to extracellular matrix (anoikis) than cells from the wild-type or heterozygous littermates. MEFs and tissues from Bit1 KO mice displayed a marked increase in Erk phosphorylation. Knocking down Bit1 expression in cultured cells resulted in increased Erk activation, and partially knocking down Erk reversed the increased anoikis resistance of Bit1 knockdown. The enhanced Erk activation was associated with decreased Erk phosphatase activity. These studies establish the physiological significance of Bit1 activity and begin to delineate a Bit1 signaling pathway that acts through Erk regulation.

A novel Flk1-TVA transgenic mouse model for gene delivery to angiogenic vasculature.

The genes that regulate the formation of blood vessels in adult tissues represent promising therapeutic targets because angiogenesis plays a role in many diseases, including cancer. We wished to develop a mouse model allowing characterization of gene function in adult angiogenic vasculature while minimizing effects on embryonic vasculature or adult quiescent vasculature. Here we describe a transgenic mouse model that allows expression of proteins in the endothelial cells of newly forming blood vessels in the adult using a selective retroviral gene delivery system. We generated transgenic mouse lines that express the TVA receptor for the RCAS avian-specific retrovirus from Flk1 gene regulatory elements that drive expression in proliferating endothelial cells. Several of these Flk1-TVA lines expressed TVA mRNA in the embryonic vasculature and TVA protein in new blood vessels growing into subcutaneous extracellular matrix implants in adult mice. In a Flk1-TVA line that was crossed with the MMTV-PyMT transgenic mammary tumor model, tumor endothelial cells also expressed the TVA protein. Furthermore, endothelial cells in extracellular matrix implants and the tumors of Flk1-TVA mice were susceptible to RCAS infection, as determined by expression of green fluorescent protein encoded by the virus. The Flk1-TVA mouse model in conjunction with the RCAS gene delivery system will be useful to study molecular mechanisms underlying adult forms of angiogenesis.