Sunish Mohanan

Implanted adipose progenitor cells as physicochemical regulators of breast cancer

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.

Glioblastoma Stem Cells Are Regulated by Interleukin-8 Signaling in a Tumoral Perivascular Niche

Glioblastoma multiforme contains a subpopulation of cancer stem-like cells (CSC) believed to underlie tumorigenesis and therapeutic resistance. Recent studies have localized CSCs in this disease adjacent to endothelial cells (EC) in what has been termed a perivascular niche, spurring investigation into the role of EC-CSC interactions in glioblastoma multiforme pathobiology. However, these studies have been limited by a lack of in vitro models of three-dimensional disease that can recapitulate the relevant conditions of the niche. In this study, we engineered a scaffold-based culture system enabling brain endothelial cells to form vascular networks. Using this system, we showed that vascular assembly induces CSC maintenance and growth in vitro and accelerates tumor growth in vivo through paracrine interleukin (IL)-8 signaling. Relative to conventional monolayers, endothelial cells cultured in this three-dimensional system not only secreted enhanced levels of IL-8 but also induced CSCs to upregulate the IL-8 cognate receptors CXCR1 and CXCR2, which collectively enhanced CSC migration, growth, and stemness properties. CXCR2 silencing in CSCs abolished the tumor-promoting effects of endothelial cells in vivo, confirming a critical role for this signaling pathway in GMB pathogenesis. Together, our results reveal synergistic interactions between endothelial cells and CSCs that promote the malignant properties of CSCs in an IL-8-dependent manner. Furthermore, our findings underscore the relevance of tissue-engineered cell culture platforms to fully analyze signaling mechanisms in the tumor microenvironment.

Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis

Obesity leads to fibrotic remodeling of mammary adipose tissue, and the resulting increase in interstitial extracellular matrix stiffness promotes breast tumor malignancy. Fat fibrosis and breast cancer One of the many risk factors for cancer is obesity—but why? Seo et al. examined the cellular, structural, and molecular changes that happen in breast tissue in obese animals and people. They found that obesity induces fibrotic remodeling of the mammary fat pad, leading to changes in extracellular matrix (ECM) mechanical properties, via myofibroblasts and adipose stem cells (ASCs), regardless of ovary function. Through altered mechanotransduction, ECM from obese mice promoted human breast cancer cell growth, as well as the growth of premalignant breast cells (those that have yet to become cancerous). Tissues from obese patients revealed more severe fibrotic remodeling around tumors and higher levels of a key mechanosignaling component, YAP/TAZ, than their lean counterparts. The authors further demonstrated that caloric restriction in obese mice decreased fibrosis in mammary fat, suggesting a therapeutic angle for obesity-related cancers. By linking tumorigenesis to the behavior of fat cells and ECM mechanics, the authors point toward new drug targets for preventing cancer progression. However, a cautionary tale also exists in the use of adipose tissue and cells for patients after mastectomy, as ASCs from obese individuals may have the capacity to promote breast cancer recurrence. Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies.

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target

BackgroundWe have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects.MethodsRNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes.ResultsWe show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 × 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45α, and Ki67.ConclusionTogether, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.

Potential Role of Peptidylarginine Deiminase Enzymes and Protein Citrullination in Cancer Pathogenesis

The peptidylarginine deiminases (PADs) are a family of posttranslational modification enzymes that catalyze the conversion of positively charged protein-bound arginine and methylarginine residues to the uncharged, nonstandard amino acid citrulline. This enzymatic activity is referred to as citrullination or, alternatively, deimination. Citrullination can significantly affect biochemical pathways by altering the structure and function of target proteins. Five mammalian PAD family members (PADs 1–4 and 6) have been described and show tissue-specific distribution. Recent reviews on PADs have focused on their role in autoimmune diseases. Here, we will discuss the potential role of PADs in tumor progression and tumor-associated inflammation. In the context of cancer, increasing clinical evidence suggests that PAD4 (and possibly PAD2) has important roles in tumor progression. The link between PADs and cancer is strengthened by recent findings showing that treatment of cell lines and mice with PAD inhibitors significantly suppresses tumor growth and, interestingly, inflammatory symptoms. At the molecular level, transcription factors, coregulators, and histones are functional targets for citrullination by PADs, and citrullination of these targets can affect gene expression in multiple tumor cell lines. Next generation isozyme-specific PAD inhibitors may have therapeutic potential to regulate both the inflammatory tumor microenvironment and tumor cell growth.

In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope

We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF. The MPME has a rigid distal tip of 3 mm in outer diameter and 4 cm in length. The image field-of-view measures 115 μm by 115 μm and was acquired at 4.1 frames/s with 75 mW illumination power at the sample. Organs were imaged after anesthetizing Sprague-Dawley rats with isofluorane gas, accessing tissues via a ventral-midline abdominal incision, and isolating the organs with tongue depressors. In vivo multiphoton images acquired from liver, kidney, and colon using this device show features similar to that of conventional histology slides, without motion artifact, in ~75% of imaged frames. To the best of our knowledge, this is the first demonstration of multiphoton imaging of unstained tissue from a live subject using a compact and flexible MPME device.

Identification of macrophage extracellular trap-like structures in mammary gland adipose tissue: a preliminary study

PAD4-mediated hypercitrullination of histone H4 arginine 3 (H4R3) has been previously found to promote the formation of Neutrophil Extracellular Traps in inflamed tissues and the resulting histone H4 citrulline 3 (H4Cit3) modification is thought to play a key role in extracellular trap (ET) formation by promoting chromatin decondensation. In addition to neutrophils, macrophages have also recently been found to generate functional extracellular traps (METs). However, a role for PADs in ET formation in macrophages has not been previously described. Transcripts for PAD2 and PAD4 are found in mature macrophages and these cells can be induced to citrullinate proteins, thus raising the possibility that PADs may play a direct role in ET formation in macrophages via histone hypercitrullination. In breast and visceral white adipose tissue from obese patients, infiltrating macrophages are often seen to surround dead adipocytes forming characteristic “crown-like structures” (CLS) and the presence of these lesions is associated with increased levels of inflammatory mediators. In light of these observations, we have initiated studies to test whether PADs are expressed in CLS macrophages and whether these macrophages might form METs. Our preliminary findings show that PAD2 (and to a lesser extent, PAD4) is expressed in both in the macrophage cell line (RAW 264.7) and in CLS lesions. Additionally, we provide evidence that macrophage-derived extracellular histones are seen around presumptive macrophages within CLS lesions and that these histones contain the H4Cit3 modification. These initial findings support our hypothesis that obesity-induced adipose tissue inflammation promotes the formation of METs within CLS lesions via PAD-mediated histone hypercitrullination. Subsequent studies are underway to further validate these findings and to investigate the role in PAD-mediated MET formation in CLS function in the mammary gland.

Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells

Peptidylarginine deiminase 4 (PAD4) is a Ca2+-dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3β (GSK3β), both in vitro and in vivo. Among other functions, GSK3β is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3β protein levels, increased TGF-β signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3β is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3β is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.

In Vivo Tibial Compression Decreases Osteolysis and Tumor Formation in a Human Metastatic Breast Cancer Model

Bone metastasis, the leading cause of breast cancer‐related deaths, is characterized by bone degradation due to increased osteoclastic activity. In contrast, mechanical stimulation in healthy individuals upregulates osteoblastic activity, leading to new bone formation. However, the effect of mechanical loading on the development and progression of metastatic breast cancer in bone remains unclear. Here, we developed a new in vivo model to investigate the role of skeletal mechanical stimuli on the development and osteolytic capability of secondary breast tumors. Specifically, we applied compressive loading to the tibia following intratibial injection of metastatic breast cancer cells (MDA‐MB231) into the proximal compartment of female immunocompromised (SCID) mice. In the absence of loading, tibiae developed histologically‐detectable tumors with associated osteolysis and excessive degradation of the proximal bone tissue. In contrast, mechanical loading dramatically reduced osteolysis and tumor formation and increased tibial cancellous mass due to trabecular thickening. These loading effects were similar to the baseline response we observed in non‐injected SCID mice. In vitro mechanical loading of MDA‐MB231 in a pathologically relevant 3D culture model suggested that the observed effects were not due to loading‐induced tumor cell death, but rather mediated via decreased expression of genes interfering with bone homeostasis. Collectively, our results suggest that mechanical loading inhibits the growth and osteolytic capability of secondary breast tumors after their homing to the bone, which may inform future treatment of breast cancer patients with advanced disease.

IL-2–inducible T-cell kinase modulates TH2-mediated allergic airway inflammation by suppressing IFN-γ in naive CD4+ T cells

BACKGROUND Asthma is a predominantly TH2 cell-dominated inflammatory disease characterized by airway inflammation and a major public health concern affecting millions of persons. The Tec family tyrosine kinase IL-2-inducible T-cell kinase (Itk) is primarily expressed in T cells and critical for the function and differentiation of TH cells. Itk(-/-) mice have a defective TH2 response and are not susceptible to allergic asthma. OBJECTIVE We sought to better understand the role of Itk signaling in TH differentiation programs and in the development and molecular pathology of allergic asthma. METHODS Using a murine model of allergic airway inflammation, we dissected the role of Itk in regulating TH cell differentiation through genetic ablation of critical genes, chromatin immunoprecipitation assays, and house dust mite-driven allergic airway inflammation. RESULTS Peripheral naive Itk(-/-) CD4(+) T cells have substantially increased transcripts and expression of the prototypic TH1 genes Eomesodermin, IFN-γ, T-box transcription factor (T-bet), and IL-12Rβ1. Removal of IFN-γ on the Itk(-/-) background rescues expression of TH2-related genes in TH cells and allergic airway inflammation in Itk(-/-) mice. Furthermore, small hairpin RNA-mediated knockdown of Itk in human peripheral blood T cells results in increased expression of mRNA for IFN-γ and T-bet and reduction in expression of IL-4. CONCLUSION Our results indicate that Itk signals suppress the expression of IFN-γ in naive CD4(+) T cells, which in a positive feed-forward loop regulates the expression of TH1 factors, such as T-bet and Eomesodermin, and suppress development of TH2 cells and allergic airway inflammation.