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Evidence link bacterial enterotoxins to apparent crypt-cell like cells (CCLCs), and Alpha Defensin 5 (DEFA5) expansion in the colonic mucosa of Crohn’s colitis disease (CC) patients. These areas of ectopic ileal metaplasia, positive for Paneth cell (PC) markers are consistent with diagnosis of CC. Retrospectively, we: 1. Identified 21 patients with indeterminate colitis (IC) between 2000–2007 and were reevaluation their final clinical diagnosis in 2014 after a followed-up for mean 8.7±3.7 (range, 4–14) years. Their initial biopsies were analyzed by DEFA5 bioassay. 2. Differentiated ulcer-associated cell lineage (UACL) analysis by immunohistochemistry (IHC) of the CC patients, stained for Mucin 6 (MUC6) and DEFA5. 3. Treated human immortalized colonic epithelial cells (NCM460) and colonoids with pure DEFA5 on the secretion of signatures after 24hr. The control colonoids were not treated. 4. Treated colonoids with/without enterotoxins for 14 days and the spent medium were collected and determined by quantitative expression of DEFA5, CCLCs and other biologic signatures. The experiments were repeated twice. Three statistical methods were used: (i) Univariate analysis; (ii) LASSO; and (iii) Elastic net. DEFA5 bioassay discriminated CC and ulcerative colitis (UC) in a cohort of IC patients with accuracy. A fit logistic model with group CC and UC as the outcome and the DEFA5 as independent variable differentiator with a positive predictive value of 96 percent. IHC staining of CC for MUC6 and DEFA5 stained in different locations indicating that DEFA5 is not co-expressed in UACL and is therefore NOT the genesis of CC, rather a secretagogue for specific signature(s) that underlie the distinct crypt pathobiology of CC. Notably, we observed expansion of signatures after DEFA5 treatment on NCM460 and colonoids cells expressed at different times, intervals, and intensity. These factors are key stem cell niche regulators leading to DEFA5 secreting CCLCs differentiation ‘the colonic ectopy ileal metaplasia formation’ conspicuously of pathogenic importance in CC.

The present studies were conducted to evaluate key serum proteins and other components that mediate anchorage-independent growth (3-D growth) of LNCaP prostate cancer cells as spheroids. The cells were cultured on ultra-low attachment plates in the absence and presence of fetuin-A and with or without extracellular vesicles. The data show that fetuin-A (alpha 2HS glycoprotein) is the serum protein that mediates 3-D growth in these cells. It does so by sequestering extracellular vesicles of various sizes on the surfaces of rounded cells that grow as spheroids. These vesicles in turn transmit growth signals such as the activation of AKT and MAP kinases in a pattern that differs from the activation of these key growth signaling pathways in adherent and spread cells growing in 2-D. In the process of orchestrating the movement and disposition of extracellular vesicles on these cells, fetuin-A is readily internalized in adhered and spread cells but remains on the surfaces of non-adherent cells. Taken together, our studies suggest the presence of distinct signaling domains or scaffolding platforms on the surfaces of prostate tumor cells growing in 3-D compared to 2-D.

The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs., Our data show that lapatinib-induced annexin A6 upregulation and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for epidermal growth factor receptor (EGFR)-expressing triple-negative breast cancer cells to overcome prolong treatment with EGFR-targeted tyrosine kinase inhibitors and could be targeted to disrupt and/or monitor the acquired resistance.

Cancer-induced hypercalcemia (CIH) is common in cancer patients with metastatic disease and in up to 30% of breast cancer cases without evidence of metastasis. During malignancy, tumor cells-including triple negative breast cancer (TNBC) cells, secrete factors such as parathyroid hormone-related protein (PTHrP) that causes the osteolysis. As cancer progresses, it becomes inevitable that the increase in tumor cell derived PTHrP and the associated persistent osteolysis, leads to a progressive increase in systemic calcium (Ca2+) or CIH. The resulting increase in circulating Ca2+ is sensed by the calcium-sensing receptor (CaSR), which not only plays a significant role in maintaining Ca2+ homeostasis but also promotes tumor cell proliferation and motility. High circulating Ca2+ is associated with aggressive tumors in premenopausal women and larger tumors in postmenopausal women, but the contribution of the CaSR remains poorly understood. Since breast cancer frequently metastasizes to Ca2+-rich skeletal tissues, the goal of this study is to gain a better understanding of the role of CaSR in the adaptation of TNBC cells to high Ca2+ and subsequent survival in Ca2+ rich environments. More than 200 mutations, including single nucleotide polymorphisms (SNPs) in exon 7 of the CaSR gene, have been described. These include the inactivating A986S CaSR at rs1801725 and Q1011E CaSR at rs1801726 with reduced sensitivity to Ca2+. Unlike SNPs at rs1801726, up to 20% of breast cancer patients with SNPs at rs1801725 may be predisposed to higher circulating Ca2+ in the course of their disease. Our preliminary data reveal that the expression level and mutational status (in exon 7) of the CaSR is cell type specific, where sustained high Ca2+ desensitizes the receptor, but promotes tumor cell growth and motility. Sustained high Ca2+ triggers growth and the expression of metastasis promoting genes including the cancer/testis (melanoma) antigen, MAGEC2 and Plasminogen Activator Inhibitor, PAI-2, potentially via the early response genes FOS/FOSB. Moreover, our preliminary data showed that the A986S SNP is associated with hypercalcemia and secondary malignancy of bone, while the Q1011E SNP is associated with osteoporosis. We hypothesize that desensitization of the CaSR by sustained high Ca2+ is critical for both the adaptation of TNBC cells to CIH, and their survival in Ca2+ rich microenvironments. To test this hypothesis, we will determine whether high Ca2+ inducible genes/proteins underlie the adaptation of TNBC cells in high Ca2+ microenvironments. We anticipate that data from this proposed study will provide novel insights into the adaptation of TNBC cells in high Ca2+ prior to metastasis. This proposed research will study how the differences in the functional status of the CaSR (A986S in Caucasians and Q1011E in African Americans) may predispose breast cancer patients to rapid progression of advanced forms of breast cancer and becoming hypercalcemic. Citation Format: Heather K. Beasley, Ky'Era Actkins, Annika Faucon, Diva Whalen, Stephen Williams, Olga Korolkova, Amos Sakwe. The role of the calcium-sensing receptor (CaSR) in the bone metastatic niche [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1567.

Actively growing tumors are often histologically associated with Ki67 positivity, while the detection of invasiveness relies on non-quantitative pathologic evaluation of mostly advanced tumors. We recently reported that reduced expression of the Ca2+-dependent membrane-binding annexin A6 (AnxA6) is associated with increased expression of the Ca2+ activated RasGRF2 (GRF2), and that the expression status of these proteins inversely influence the growth and motility of triple negative breast cancer (TNBC) cells. Here, we establish that the reciprocal expression of AnxA6 and GRF2 is at least in part, dependent on inhibition of non-selective Ca2+ channels in AnxA6-low but not AnxA6-high TNBC cells. Immunohistochemical staining of breast cancer tissues revealed that compared to non-TNBC tumors, TNBC tumors express lower levels of AnxA6 and higher Ki67 expression. GRF2 expression levels strongly correlated with high Ki67 in pretreatment biopsies from patients with residual disease and with residual tumor size following chemotherapy. Elevated AnxA6 expression more reliably identified patients who responded to chemotherapy, while low AnxA6 levels were significantly associated with shorter distant relapse-free survival. Finally, the reciprocal expression of AnxA6 and GRF2 can delineate GRF2-low/AnxA6-high invasive from GRF2-high/AnxA6-low rapidly growing TNBCs. These data suggest that AnxA6 may be a reliable biomarker for distant relapse-free survival and response of TNBC patients to chemotherapy, and that the reciprocal expression of AnxA6 and GRF2 can reliably delineate TNBCs into rapidly growing and invasive subsets which may be more relevant for subset-specific therapeutic interventions.

Inability to distinguish Crohn's colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn's colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn's colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn's colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohn's disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn's colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn's colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn's colitis.

Inability to distinguish Crohn's colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn's colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn's colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn's colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohn's disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn's colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn's colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn's colitis.

We analyzed inflamed mucosal/submucosal layers of ulcerative colitis (UC = 63) and Crohn's colitis (CC = 50), and unexpectedly, we unveiled a pool of free hemoglobin alpha (Hb-α) chain. Patients with colitides have increased reactive oxidative stress (ROS), DNA oxidation products, free iron in mucosa, in preneoplastic, and in colitis-cancers and increased risks of developing colorectal cancer. All inflammatory bowel disease-related colorectal cancer lesions are found in segments with colitis. Linking this information, we investigated whether free Hb-α is key transformational stepping that increases colitis-related colorectal cancer vulnerability.UC/CC samples were profiled using matrix-assisted laser desorption/ionization mass spectrometry; protein identification was made by liquid chromatography. Diverticulitis was used as control (Ctrl). The presence of Hb(n) (n = α, β, or hemin)/Hb was validated by Western blotting and immunohistochemistry. We tested for DNA damage (DNAD) by exposing normal colonic epithelial cell line, NCM460, to 10 μM and 100 μM of Hb(n)/Hb, individually for 2, 6, and 12 hours. Quantification of Hb-α staining was done by Nikon Elements Advance Research Analysis software. ROS was measured by the production of 8-OHdG. DNAD was assessed by Comet assay. Colonic tissue homogenate antioxidants Nrf2-, CAT-, SOD-, and GPx-expressions were analyzed densitometrically/normalized by β-actin.Immunohistochemistry of CC/UC mucosal/submucosal compartments stained strongly positive for Hb-α and significantly higher versus Ctrl. NCM460 exposed to Hb(n)/Hb exhibited steadily increasing ROS and subsequent DNAD. DNAD was higher in 10 μM than 100 μM in Hb-β/hemin the first 2 hours then plateaued followed by DNAD repair. This may be likely due to apoptosis in the later concentration. Nrf2 enzyme activities among UC, CC, and ulcerative colitis-associated colon cancer (UCAC) were observed impaired in all inflammatory bowel disease subjects. Decreased levels of Nrf2 among patients with UC versus patients with CC with active disease were insignificant as well as versus Ctrls but significantly lower in UCAC versus Ctrl. SOD was decreased in UC and UCAC and GPx in CC but statistically not significant. Comparing CC versus UC, SOD was significantly lower in CC (P0.05). CAT was observed increased among patients with CC/UC/UCAC and GPx in UC and UCAC versus Ctrl, respectively, and significantly increased in CC versus Ctrl (P0.01).In the colitides, mucosal/submucosal tissue microenvironments demonstrated pool of free Hb-α chain. In vitro exposure of NCM460 cells to Hb(n)/Hb induced ROS and DNAD. Toxic effect of free Hb-α, in colonic epithelial cells, is therefore through production of ROS formation modulated by impairment of antioxidant effects. Targeting reduction-oxidation-sensitive pathways and transcription factors may offer options for inflammatory bowel disease-management and colitis-related cancer prevention.

Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett's metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERD-associated tumorigenesis.

BackgroundAs accessible diagnostic approaches fail to differentiate between ulcerative colitis (UC) and Crohn's colitis (CC) in one-third of patients with predominantly colonic inflammatory bowel disease (IBD), leading to inappropriate therapy, we aim to investigate the serum cytokine levels in these patients in search of molecular biometric markers delineating UC from CC.MethodsWe measured 38 cytokines, chemokines, and growth factors using magnetic-bead-based multiplex immunoassay in 25 UC patients, 28 CC patients, and 30 controls. Our results are compared with those from a review of current literature regarding advances in serum cytokine profiles and associated challenges preventing their use for diagnostic/prognostic purposes.ResultsUnivariate analysis showed statistically significant increases of eotaxin, GRO, and TNF-α in UC patients compared to controls (Ctrl); interferon γ, interleukin (IL)-6, and IL-7 in CC group compared to Ctrl; and IL-8 in both UC and CC versus Ctrl. No cytokines were found to be different between UC and CC. A generalized linear model identified combinations of cytokines, allowing the identification of UC and CC patients, with area under the curve (AUC) = 0.936, as determined with receiver operating characteristic (ROC) analysis.ConclusionsThe current knowledge available about circulating cytokines in IBD is often contradictory. The development of an evidence-based tool using cytokines for diagnostic accuracy is still preliminary.

The role of AnxA6 in breast cancer and in particular, the mechanisms underlying its contribution to tumor cell growth and/or motility remain poorly understood. In this study, we established the tumor suppressor function of AnxA6 in triple negative breast cancer (TNBC) cells by showing that loss of AnxA6 is associated with early onset and rapid growth of xenograft TNBC tumors in mice. We also identified the Ca2+ activated RasGRF2 as an effector of AnxA6 mediated TNBC cell growth and motility. Activation of Ca2+ mobilizing oncogenic receptors such as epidermal growth factor receptor (EGFR) in TNBC cells or pharmacological stimulation of Ca2+ influx led to activation, subsequent degradation and altered effector functions of RasGRF2. Inhibition of Ca2+ influx or overexpression of AnxA6 blocked the activation/degradation of RasGRF2. We also show that AnxA6 acts as a scaffold for RasGRF2 and Ras proteins and that its interaction with RasGRF2 is modulated by GTP and/or activation of Ras proteins. Meanwhile, down-regulation of RasGRF2 and treatment of cells with the EGFR-targeted tyrosine kinase inhibitor (TKI) lapatinib strongly attenuated the growth of otherwise EGFR-TKI resistant AnxA6 high TNBC cells. These data not only suggest that AnxA6 modulated Ca2+ influx and effector functions of RasGRF2 underlie at least in part, the AnxA6 mediated TNBC cell growth and/or motility, but also provide a rationale to target Ras-driven TNBC with EGFR targeted therapies in combination with inhibition of RasGRF2.

This method makes it possible to measure the fluorescence of a DNA probe in cells with known division number and targeted surface antigen. In fact, this method is a combination or consistent application of three other methods: cell tracking by vital dye, surface immunophenotyping, and flow-FISH. The idea in developing this method was to study telomere length changes in cells with known surface antigen after every new cell division. First, the in vitro cell culturing and staining with CFSE vital dye are performed. Then, cells are stained with surface MAbs labeled with biotin, followed by incubation with streptavidin-labeled fluorochrome. After that, cells are fixed with BS(3) reagent followed by the flow-FISH procedure with PNA-probe complementary to telomere DNA repeats. Finally, in one tube, it is possible to determine telomere length in surface antigen-labeled cells that have made the exact same number of divisions after incubation.