Reprogramming of breast tumor-associated macrophages with modulation of arginine metabolism.

HER2+ breast tumors have abundant immune-suppressive cells, including M2-type tumor associated macrophages (TAMs). While TAMs consist of the immune-stimulatory M1-type and immune-suppressive M2-type, M1/M2-TAM ratio is reduced in immune-suppressive tumors, contributing to their immunotherapy refractoriness. M1 vs. M2-TAM formation depends on differential arginine metabolism, where M1-TAMs convert arginine to nitric oxide (NO) and M2-TAMs convert arginine to polyamines (PAs). We hypothesize that such distinct arginine metabolism in M1- vs M2-TAMs is attributed to different availability of BH4 (NO synthase cofactor) and that its replenishment would reprogram M2-TAMs to M1-TAMs. Recently, we reported that sepiapterin (SEP), the endogenous BH4 precursor, elevates the expression of M1-TAM markers within HER2+ tumors. Here, we show that SEP restores BH4 levels in M2-TAMs, which then redirects arginine metabolism to NO synthesis and converts M2-TAMs to M1-TAMs. The reprogrammed TAMs exhibit full-fledged capabilities of antigen presentation and induction of effector T cells to trigger immunogenic cell death of HER2+ cancer cells. This study substantiates the utility of SEP in metabolic shift of HER2+ breast tumor microenvironment as a novel immunotherapeutic strategy.

Reduced S-nitrosylation of TGFß1 elevates its binding affinity towards the receptor and promotes fibrogenic signaling in the breast.

Transforming Growth Factor ß (TGFß) is a pleiotropic cytokine closely linked to tumors. TGFß is often elevated in precancerous breast lesions in association with epithelial-to-mesenchymal transition (EMT), indicating its contribution to precancerous progression. We previously reported that basal nitric oxide (NO) levels declined along with breast cancer progression. We then pharmacologically inhibited NO production in healthy mammary glands of wild-type mice and found that this induced precancerous progression accompanied by desmoplasia and upregulation of TGFß activity. In the present study, we tested our hypothesis that NO directly S-nitrosylates (forms an NO-adduct at a cysteine residue) TGFß to inhibit the activity, whereas the reduction of NO denitrosylates TGFß and de-represses the activity. We introduced mutations to three C-terminal cysteines of TGFß1 which were predicted to be S-nitrosylated. We found that these mutations indeed impaired S-nitrosylation of TGFß1 and shifted the binding affinity towards the receptor from the latent complex. Furthermore, in silico structural analyses predicted that these S-nitrosylation-defective mutations strengthen the dimerization of mature protein, whereas S-nitrosylation-mimetic mutations weaken the dimerization. Such differences in dimerization dynamics of TGFß1 by denitrosylation/S-nitrosylation likely account for the shift of the binding affinities towards the receptor vs. latent complex. Our findings, for the first time, unravel a novel mode of TGFß regulation based on S-nitrosylation or denitrosylation of the protein.

Unraveling Therapeutic Opportunities and the Diagnostic Potential of microRNAs for Human Lung Cancer.

Lung cancer is a major public health problem and a leading cause of cancer-related deaths worldwide. Despite advances in treatment options, the five-year survival rate for lung cancer patients remains low, emphasizing the urgent need for innovative diagnostic and therapeutic strategies. MicroRNAs (miRNAs) have emerged as potential biomarkers and therapeutic targets for lung cancer due to their crucial roles in regulating cell proliferation, differentiation, and apoptosis. For example, miR-34a and miR-150, once delivered to lung cancer via liposomes or nanoparticles, can inhibit tumor growth by downregulating critical cancer promoting genes. Conversely, miR-21 and miR-155, frequently overexpressed in lung cancer, are associated with increased cell proliferation, invasion, and chemotherapy resistance. In this review, we summarize the current knowledge of the roles of miRNAs in lung carcinogenesis, especially those induced by exposure to environmental pollutants, namely, arsenic and benzopyrene, which account for up to 1/10 of lung cancer cases. We then discuss the recent advances in miRNA-based cancer therapeutics and diagnostics. Such information will provide new insights into lung cancer pathogenesis and innovative diagnostic and therapeutic modalities based on miRNAs.

Changes in Expression of Tumor Suppressor Gene RKIP Impact How Cancers Interact with Their Complex Environment.

Tumor microenvironment (TME) is the immediate environment where cancer cells reside in a tumor. It is composed of multiple cell types and extracellular matrix. Microenvironments can be restrictive or conducive to the progression of cancer cells. Initially, microenvironments are suppressive in nature. Stepwise accumulation of mutations in oncogenes and tumor suppressor genes enables cancer cells to acquire the ability to reshape the microenvironment to advance their growth and metastasis. Among the many genetic events, the loss-of-function mutations in tumor suppressor genes play a pivotal role. In this review, we will discuss the changes in TME and the ramifications on metastasis upon altered expression of tumor metastasis suppressor gene RKIP in breast cancer cells.

Novel 3D Flipwell system that models gut mucosal microenvironment for studying interactions between gut microbiota, epithelia and immunity.

Gut mucosa consists of stratified layers of microbes, semi-permeable mucus, epithelium and stroma abundant in immune cells. Although tightly regulated, interactions between gut commensals and immune cells play indispensable roles in homeostasis and cancer pathogenesis in the body. Thus, there is a critical need to develop a robust model for the gut mucosal microenvironment. Here, we report our novel co-culture utilizing 3D Flipwell system for establishing the stratified layers of discrete mucosal components. This method allows for analyzing synchronous effects of test stimuli on gut bacteria, mucus, epithelium and immune cells, as well as their crosstalks. In the present report, we tested the immuno-stimulatory effects of sepiapterin (SEP, the precursor of the cofactor of nitric oxide synthase (NOS)-BH4) on the gut mucosal community. We previously reported that SEP effectively reprogrammed tumor-associated macrophages and inhibited breast tumor cell growth. In our co-cultures, SEP largely promoted mucus integrity, bacterial binding, and M1-like polarization of macrophages. Conversely, these phenomena were absent in control-treated cultures. Our results demonstrate that this novel co-culture may serve as a robust in vitro system to recapitulate the effects of pharmacological agents on the gut mucosal microenvironment, and could potentially be expanded to test the effects outside the gut.

CD36+ Fibroblasts Secrete Protein Ligands That Growth-Suppress Triple-Negative Breast Cancer Cells While Elevating Adipogenic Markers for a Model of Cancer-Associated Fibroblast.

Tumor and stroma coevolve to facilitate tumor growth. Hence, effective tumor therapeutics would not only induce growth suppression of tumor cells but also revert pro-tumor stroma into anti-tumoral type. Previously, we showed that coculturing triple-negative or luminal A breast cancer cells with CD36+ fibroblasts (FBs) in a three-dimensional extracellular matrix induced their growth suppression or phenotypic reversion, respectively. Then, we identified SLIT3, FBLN-1, and PENK as active protein ligands secreted from CD36+ FBs that induced growth suppression of MDA-MB-231 breast cancer cells and determined their minimum effective concentrations. Here, we have expanded our analyses to include additional triple-negative cancer cell lines, BT549 and Hs578T, as well as HCC1937 carrying a BRCA1 mutation. We show that the ectopic addition of each of the three ligands to cancer-associated fibroblasts (CAFs) elevates the expression of CD36, as well as the adipogenic marker FABP4. Lastly, we show that an agonist antibody for one of the PENK receptors induces growth suppression of all cancer cell lines tested but not for non-transformed MCF10A cells. These results clearly suggest that proteins secreted from CD36+ FBs induce not only growth suppression of tumor cells through binding the cognate receptors but also increasing adipogenic markers of CAFs to reprogram tumor stroma.

Fibrous stroma: Driver and passenger in cancer development.

Cumulative evidence shows that fibrogenic stroma and stiff extracellular matrix (ECM) not only result from tumor growth but also play pivotal roles in cellular transformation and tumor initiation. This emerging concept may largely account for the increased cancer risk associated with environmental fibrogenic agents, such as asbestos and silica, and with chronic conditions that are fibrogenic, such as obesity and diabetes. It may also contribute to poor outcomes in patients treated with certain chemotherapeutics that can promote fibrosis, such as bleomycin and methotrexate. Although the mechanistic details of this phenomenon are still being unraveled, we provide an overview of the experimental evidence linking fibrogenic stroma and tumor initiation. In this Review, we will summarize the causes and consequences of fibrous stroma and how this stromal cue is transmitted to the nuclei of parenchymal cells through a physical continuum from the ECM to chromatin, as well as ECM-dependent biochemical signaling that contributes to cellular transformation.

Protein Ligands in the Secretome of CD36+ Fibroblasts Induce Growth Suppression in a Subset of Breast Cancer Cell Lines.

Reprogramming the tumor stroma is an emerging approach to circumventing the challenges of conventional cancer therapies. This strategy, however, is hampered by the lack of a specific molecular target. We previously reported that stromal fibroblasts (FBs) with high expression of CD36 could be utilized for this purpose. These studies are now expanded to identify the secreted factors responsible for tumor suppression. Methodologies included 3D colonies, fluorescent microscopy coupled with quantitative techniques, proteomics profiling, and bioinformatics analysis. The results indicated that the conditioned medium (CM) of the CD36+ FBs caused growth suppression via apoptosis in the triple-negative cell lines of MDA-MB-231, BT549, and Hs578T, but not in the ERBB2+ SKBR3. Following the proteomics and bioinformatic analysis of the CM of CD36+ versus CD36- FBs, we determined KLF10 as one of the transcription factors responsible for growth suppression. We also identified FBLN1, SLIT3, and PENK as active ligands, where their minimum effective concentrations were determined. Finally, in MDA-MB-231, we showed that a mixture of FBLN1, SLIT3, and PENK could induce an amount of growth suppression similar to the CM of CD36+ FBs. In conclusion, our findings suggest that these ligands, secreted by CD36+ FBs, can be targeted for breast cancer treatment.

Loss of Nitric Oxide Induces Fibrogenic Response in Organotypic 3D Co-Culture of Mammary Epithelia and Fibroblasts-An Indicator for Breast Carcinogenesis.

Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial-mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer.

S-Nitrosylation in Tumor Microenvironment.

S-nitrosylation is a selective and reversible post-translational modification of protein thiols by nitric oxide (NO), which is a bioactive signaling molecule, to exert a variety of effects. These effects include the modulation of protein conformation, activity, stability, and protein-protein interactions. S-nitrosylation plays a central role in propagating NO signals within a cell, tissue, and tissue microenvironment, as the nitrosyl moiety can rapidly be transferred from one protein to another upon contact. This modification has also been reported to confer either tumor-suppressing or tumor-promoting effects and is portrayed as a process involved in every stage of cancer progression. In particular, S-nitrosylation has recently been found as an essential regulator of the tumor microenvironment (TME), the environment around a tumor governing the disease pathogenesis. This review aims to outline the effects of S-nitrosylation on different resident cells in the TME and the diverse outcomes in a context-dependent manner. Furthermore, we will discuss the therapeutic potentials of modulating S-nitrosylation levels in tumors.

Correction of arginine metabolism with sepiapterin-the precursor of nitric oxide synthase cofactor BH4-induces immunostimulatory-shift of breast cancer.

Immunotherapy is a first-line treatment for many tumor types. However, most breast tumors are immuno-suppressive and only modestly respond to immunotherapy. We hypothesized that correcting arginine metabolism might improve the immunogenicity of breast tumors. We tested whether supplementing sepiapterin, the precursor of tetrahydrobiopterin (BH4)-the nitric oxide synthase (NOS) cofactor-redirects arginine metabolism from the pathway synthesizing polyamines to that of synthesizing nitric oxide (NO) and make breast tumors more immunogenic. We showed that sepiapterin elevated NO but lowered polyamine levels in tumor cells, as well as in tumor-associated macrophages (TAMs). This not only suppressed tumor cell proliferation, but also induced the conversion of TAMs from the immuno-suppressive M2-type to immuno-stimulatory M1-type. Furthermore, sepiapterin abrogated the expression of a checkpoint ligand, PD-L1, in tumors in a STAT3-dependent manner. This is the first study which reveals that supplementing sepiapterin normalizes arginine metabolism, improves the immunogenicity and inhibits the growth of breast tumor cells.

S-Nitrosylation: An Emerging Paradigm of Redox Signaling.

Nitric oxide (NO) is a highly reactive molecule, generated through metabolism of L-arginine by NO synthase (NOS). Abnormal NO levels in mammalian cells are associated with multiple human diseases, including cancer. Recent studies have uncovered that the NO signaling is compartmentalized, owing to the localization of NOS and the nature of biochemical reactions of NO, including S-nitrosylation. S-nitrosylation is a selective covalent post-translational modification adding a nitrosyl group to the reactive thiol group of a cysteine to form S-nitrosothiol (SNO), which is a key mechanism in transferring NO-mediated signals. While S-nitrosylation occurs only at select cysteine thiols, such a spatial constraint is partially resolved by transnitrosylation, where the nitrosyl moiety is transferred between two interacting proteins to successively transfer the NO signal to a distant location. As NOS is present in various subcellular locales, a stress could trigger concerted S-nitrosylation and transnitrosylation of a large number of proteins involved in divergent signaling cascades. S-nitrosylation is an emerging paradigm of redox signaling by which cells confer protection against oxidative stress.

Reduced Basal Nitric Oxide Production Induces Precancerous Mammary Lesions via ERBB2 and TGFß.

One third of newly diagnosed breast cancers in the US are early-stage lesions. The etiological understanding and treatment of these lesions have become major clinical challenges. Because breast cancer risk factors are often linked to aberrant nitric oxide (NO) production, we hypothesized that abnormal NO levels might contribute to the formation of early-stage breast lesions. We recently reported that the basal level of NO in the normal breast epithelia plays crucial roles in tissue homeostasis, whereas its reduction contributes to the malignant phenotype of cancer cells. Here, we show that the basal level of NO in breast cells plummets during cancer progression due to reduction of the NO synthase cofactor, BH4, under oxidative stress. Importantly, pharmacological deprivation of NO in prepubertal to pubertal animals stiffens the extracellular matrix and induces precancerous lesions in the mammary tissues. These lesions overexpress a fibrogenic cytokine, TGFß, and an oncogene, ERBB2, accompanied by the occurrence of senescence and stem cell-like phenotype. Consistently, normalization of NO levels in precancerous and cancerous breast cells downmodulates TGFß and ERBB2 and ameliorates their proliferative phenotype. This study sheds new light on the etiological basis of precancerous breast lesions and their potential prevention by manipulating the basal NO level.

Reconstituting Breast Tissue with Organotypic Three-dimensional Co-culture of Epithelial and Stromal Cells in Discontinuous Extracellular Matrices.

Co-culture systems utilizing reconstituted or synthetic extracellular matrix (ECM) and micropatterning techniques have enabled the reconstruction of surface epithelial tissues. This technique has been utilized in the regeneration, disease modeling and drug screening of the surface epithelia, such as the skin and esophagus. On the other hand, the reconstruction of glandular epithelia would require more intricate ECM organizations. Here we describe a protocol for a novel three-dimensional organotypic co-culture system for the reconstruction of mammary glands that utilizes the discontinuous ECM. In this technique, primary mammary fibroblasts first establish a layer of the connective tissue rich in collagen I. Then, mammary epithelial cells form acinar structures, the functional glandular units, within the laminin-rich basement membrane embedded in the connective tissue. This method allows for the regeneration of the in vivo-like architecture of mammary glands and could be utilized for monitoring the real-time response of mammary glands to drug treatment.

Transient external force induces phenotypic reversion of malignant epithelial structures via nitric oxide signaling.

Non-malignant breast epithelial cells cultured in three-dimensional laminin-rich extracellular matrix (lrECM) form well organized, growth-arrested acini, whereas malignant cells form continuously growing disorganized structures. While the mechanical properties of the microenvironment have been shown to contribute to formation of tissue-specific architecture, how transient external force influences this behavior remains largely unexplored. Here, we show that brief transient compression applied to single malignant breast cells in lrECM stimulated them to form acinar-like structures, a phenomenon we term 'mechanical reversion.' This is analogous to previously described phenotypic 'reversion' using biochemical inhibitors of oncogenic pathways. Compression stimulated nitric oxide production by malignant cells. Inhibition of nitric oxide production blocked mechanical reversion. Compression also restored coherent rotation in malignant cells, a behavior that is essential for acinus formation. We propose that external forces applied to single malignant cells restore cell-lrECM engagement and signaling lost in malignancy, allowing them to reestablish normal-like tissue architecture.

Laminin signals initiate the reciprocal loop that informs breast-specific gene expression and homeostasis by activating NO, p53 and microRNAs.

How mammalian tissues maintain their architecture and tissue-specificity is poorly understood. Previously, we documented both the indispensable role of the extracellular matrix (ECM) protein, laminin-111 (LN1), in the formation of normal breast acini, and the phenotypic reversion of cancer cells to acini-like structures in 3-dimensional (3D) gels with inhibitors of oncogenic pathways. Here, we asked how laminin (LN) proteins integrate the signaling pathways necessary for morphogenesis. We report a surprising reciprocal circuitry comprising positive players: laminin-5 (LN5), nitric oxide (NO), p53, HOXD10 and three microRNAs (miRNAs) - that are involved in the formation of mammary acini in 3D. Significantly, cancer cells on either 2-dimensional (2D) or 3D and non-malignant cells on 2D plastic do not produce NO and upregulate negative players: NFκB, EIF5A2, SCA1 and MMP-9 - that disrupt the network. Introducing exogenous NO, LN5 or individual miRNAs to cancer cells reintegrates these pathways and induces phenotypic reversion in 3D. These findings uncover the essential elements of breast epithelial architecture, where the balance between positive- and negative-players leads to homeostasis.

Basal S-Nitrosylation Is the Guardian of Tissue Homeostasis.

Recent studies have uncovered that nitric oxide (NO) signaling is largely conducted by S-nitrosylation, involving >3000 proteins. The nitrosyl group could then travel further by transnitrosylation or be secreted, enabling regulation of the whole tissue. A subset of proteins are constitutively S-nitrosylated, playing roles in the regulation of tissue homeostasis.

Pathways Involved in Formation of Mammary Organoid Architecture Have Keys to Understanding Drug Resistance and to Discovery of Druggable Targets.

Signals from the extracellular matrix (ECM) are received at the cell surface receptor, transmitted to the cytoskeletons, and transferred to the nucleus and chromatin for tissue- and context-specific gene expression. Cells, in return, modulate the cell shape and ECM, allowing for the maintenance of tissue homeostasis as well as for coevolution and adaptation to the environmental signals. We postulated the existence of dynamic and reciprocal interactions between the ECM and the nucleus more than three decades ago, but now these pathways have been proven experimentally thanks to the advances in imaging and cell/molecular biology techniques. In this review, we will introduce some of our recent work that has validated the critical roles of the three-dimensional (3D) tissue architecture in determining mammary biology, therapeutic response, and druggable targets. We describe a novel screen based on reversion of the malignant phenotype in 3D assays. We will also summarize our recent discoveries of the integration of feedback signaling for mammary acinar formation and phenotypic reversion of tumor cells in the LrECM. Lastly, we will introduce our exciting discovery of the physical linkages between the cell surface and cytofibers within a tunnel deep inside of the nucleus, enabling interaction with nuclear lamin and SUN proteins.

NFkB disrupts tissue polarity in 3D by preventing integration of microenvironmental signals.

The microenvironment of cells controls their phenotype, and thereby the architecture of the emerging multicellular structure or tissue. We have reported more than a dozen microenvironmental factors whose signaling must be integrated in order to effect an organized, functional tissue morphology. However, the factors that prevent integration of signaling pathways that merge form and function are still largely unknown. We have identified nuclear factor kappa B (NFkB) as a transcriptional regulator that disrupts important microenvironmental cues necessary for tissue organization. We compared the gene expression of organized and disorganized epithelial cells of the HMT-3522 breast cancer progression series: the non-malignant S1 cells that form polarized spheres ('acini'), the malignant T4-2 cells that form large tumor-like clusters, and the 'phenotypically reverted' T4-2 cells that polarize as a result of correction of the microenvironmental signaling. We identified 180 genes that display an increased expression in disorganized compared to polarized structures. Network, GSEA and transcription factor binding site analyses suggested that NFkB is a common activator for the 180 genes. NFkB was found to be activated in disorganized breast cancer cells, and inhibition of microenvironmental signaling via EGFR, beta1 integrin, MMPs, or their downstream signals suppressed its activation. The postulated role of NFkB was experimentally verified: Blocking the NFkB pathway with a specific chemical inhibitor or shRNA induced polarization and inhibited invasion of breast cancer cells in 3D cultures. These results may explain why NFkB holds promise as a target for therapeutic intervention: Its inhibition can reverse the oncogenic signaling involved in breast cancer progression and integrate the essential microenvironmental control of tissue architecture.

ATM suppresses SATB1-induced malignant progression in breast epithelial cells.

SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.