Overall survival of early-stage breast cancer patients is similar for those who undergo breast-conserving therapy (BCT) and mastectomy; however, 10% to 15% of women undergoing BCT suffer ipsilateral ...breast tumor recurrence. The risk of recurrence may vary with breast cancer subtype. Understanding the gene expression of the cancer-adjacent tissue and the stromal response to specific tumor subtypes is important for developing clinical strategies to reduce recurrence risk.
We utilized two independent datasets to study gene expression data in cancer-adjacent tissue from invasive breast cancer patients. Complementary in vitro cocultures were used to study cell-cell communication between fibroblasts and specific breast cancer subtypes.
Our results suggest that intrinsic tumor subtypes are reflected in histologically normal cancer-adjacent tissue. Gene expression of cancer-adjacent tissues shows that triple-negative (Claudin-low or basal-like) tumors exhibit increased expression of genes involved in inflammation and immune response. Although such changes could reflect distinct immune populations present in the microenvironment, altered immune response gene expression was also observed in cocultures in the absence of immune cell infiltrates, emphasizing that these inflammatory mediators are secreted by breast-specific cells. In addition, although triple-negative breast cancers are associated with upregulated immune response genes, luminal breast cancers are more commonly associated with estrogen-response pathways in adjacent tissues.
Specific characteristics of breast cancers are reflected in the surrounding histologically normal tissue. This commonality between tumor and cancer-adjacent tissue may underlie second primaries and local recurrences.
Biomarkers derived from cancer-adjacent tissue may be helpful in defining personalized surgical strategies or in predicting recurrence risk.
ABSTRACT
Insulin‐like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used ...Sox9‐enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9‐EGFPLow) and reserve/facultative ISCs (Sox9‐EGFPHigh) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high‐dose radiation‐induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini‐pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9‐EGFPLow ISCs and percentage of these cells in M‐phase. IGF1 increased percentages of Sox9‐EGFPHigh ISCs inS‐phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9‐EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9‐EGFPHigh facultative ISCs but not Sox9‐EGFPLow actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.—Van Landeghem, L., Santoro, M. A., Mah, A. T., Krebs, A. E., Dehmer, J. J., McNaughton, K. K., Helmrath, M.A., Magness, S.T., Lund, P. K. IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations. FASEB J. 29, 2828‐2842 (2015). www.fasebj.org
It is widely thought that pregnancy reduces breast cancer risk, but this lacks consideration of breast cancer subtypes. While a full term pregnancy reduces risk for estrogen receptor positive (ER+) ...and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC) subtype. Basal-like subtypes represent less than 10% of breast cancers and are highly aggressive, affecting primarily young, African American women. Our previous work demonstrated that high fat diet-induced obesity in nulliparous mice significantly blunted latency in C3(1)-TAg mice, a model of BBC, potentially through the hepatocyte growth factor (HGF)/c-Met oncogenic pathway. Experimental studies have examined parity and obesity individually, but to date, the joint effects of parity and obesity have not been studied. We investigated the role of obesity in parous mice on BBC. Parity alone dramatically blunted tumor latency compared to nulliparous controls with no effects on tumor number or growth, while obesity had only a minor role in further reducing latency. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated by obesity. Plasma IL-6 was also significantly elevated by obesity in parous mice. We have previously reported a potential role for stromal-derived hepatocyte growth factor (HGF) via its cognate receptor c-Met in the etiology of obesity-induced BBC tumor onset and in both human and murine primary coculture models of BBC-aggressiveness. Obesity-associated c-Met concentrations were 2.5-fold greater in normal mammary glands of parous mice. Taken together, our studies demonstrate that, parity in C3(1)-TAg mice dramatically reduced BBC latency compared to nulliparous mice. In parous mice, c-Met is regulated by obesity in unaffected mammary gland and is associated with tumor onset. C3(1)-TAg mice recapitulate epidemiologic findings such that parity drives increased BBC risk and potential microenvironmental alterations in c-Met signaling may play a role in etiology.
Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their ...functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.
In breast tumors, it is well established that intratumoral angiogenesis is crucial for malignant progression, but little is known about the vascular characteristics of extratumoral, cancer-adjacent ...breast. Genome-wide transcriptional data suggest that extratumoral microenvironments may influence breast cancer phenotypes; thus, histologic features of cancer-adjacent tissue may also have clinical implications. To this end, we developed a digital algorithm to quantitate vascular density in approximately 300 histologically benign tissue specimens from breast cancer patients enrolled in the UNC Normal Breast Study (NBS). Specimens were stained for CD31, and vascular content was compared to demographic variables, tissue composition metrics, and tumor molecular features. We observed that the vascular density of cancer-adjacent breast was significantly higher in older and obese women, and was strongly associated with breast adipose tissue content. Consistent with observations that older and heavier women experience higher frequencies of ER+ disease, higher extratumoral vessel density was also significantly associated with positive prognostic tumor features such as lower stage, negative nodal status, and smaller size (<2 cm). These results reveal biological relationships between extratumoral vascular content and body size, breast tissue composition, and tumor characteristics, and suggest biological plausibility for the relationship between weight gain (and corresponding breast tissue changes) and breast cancer progression.
•Cancer-adjacent breast vessel density is positively associated with age and obesity.•Cancer-adjacent breast vessel density strongly varies with tissue composition.•Breast vessel density is associated with positive prognostic tumor features.•Tissue composition influences breast vessel density-tumor phenotype associations.
Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is activated by reduced endothelial shear stress and stimulates smooth muscle cell proliferation in vitro. Moreover, HB-EGF ...is augmented at sites of intimal hyperplasia and atherosclerosis, conditions favored by low/disturbed shear stress. We thus tested whether HB-EGF contributes to low flow-induced negative hypertrophic remodeling (FINR) of a mouse carotid artery. Blood flow was surgically decreased in the left and increased in the right common carotid arteries. After 21 days, the left carotid artery exhibited lumen narrowing, thickening of intima-media and adventitia, and increased circumference that were inhibited by approximately 50% in HB-EGF(+/-) and approximately 90% in HB-EGF(-/-) mice. FINR was also inhibited by the EGF receptor inhibitor AG1478. In contrast, eutrophic outward remodeling of the right carotid artery was unaffected in HB-EGF(+/-) and HB-EGF(-/-) mice, nor by AG1478. FINR-induced proliferation and leukocyte accumulation were reduced in HB-EGF(-/-). FINR was associated with increased reactive oxygen species, increased expression of pro-HB-EGF and tumor necrosis factor alpha-converting enzyme (pro-HB-EGF sheddase), increased phosphorylation of EGF receptor and extracellular signal-regulated kinase 1/2, and increased nuclear factor kappaB activity. Apocynin and deletion of p47(phox) inhibited FINR, whereas deletion of HB-EGF abolished nuclear factor kappaB activation in smooth muscle cells. These findings suggest that HB-EGF signaling is required for low flow-induced hypertrophic remodeling and may participate in vascular wall disease and remodeling.
A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic ...epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24(+) fraction that contained the majority of Lgr5-EGFP(+) putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166 in the CD24(+)EpCAM(+) fraction but also showed enrichment of quiescent CESC markers leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert). We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies.
Epidemiologic studies demonstrate that obesity is associated with an aggressive subtype of breast cancer called basal-like breast cancer (BBC). Using the C3(1)-TAg murine model of BBC, we previously ...demonstrated that mice displayed an early onset of tumors when fed obesogenic diets in the adult window of susceptibility. Obesity was also shown to elevate mammary gland expression and activation of hepatocyte growth factor (HGF)/c-Met compared to lean controls, a pro-tumorigenic pathway associated with BBC in patients. Epidemiologic studies estimate that weight loss could prevent a large proportion of BBC. We sought to investigate whether weight loss in adulthood prior to tumor onset would protect mice from accelerated tumorigenesis observed in obese mice. Using a life-long model of obesity, C3(1)-TAg mice were weaned onto and maintained on an obesogenic high-fat diet. Obese mice displayed significant elevations in tumor progression, but not latency or burden. Tumor progression was significantly reversed when obese mice were induced to lose weight by switching to a control low-fat diet prior to tumor onset compared to mice maintained on obesogenic diet. We investigated the HGF/c-Met pathway known to regulate tumorigenesis. Importantly, HGF/c-Met expression in normal mammary glands and c-Met in tumors was elevated with obesity and was significantly reversed with weight loss. Changes in tumor growth could not be explained by measures of HGF action including phospho-AKT or phospho-S6. Other mediators associated with oncogenesis such as hyperinsulinemia and a high leptin:adiponectin ratio were elevated by obesity and reduced with weight loss. In sum, weight loss significantly blunted the obesity-responsive pro-tumorigenic HGF/c-Met pathway and improved several metabolic risk factors associated with BBC, which together may have contributed to the dramatic reversal of obesity-driven tumor progression. Future research aims to evaluate the role of obesity and the HGF/c-Met pathway in basal-like breast cancer progression.
Obesity is associated with basal-like breast cancer (BBC), an aggressive breast cancer subtype. The objective of this study was to determine whether obesity promotes BBC onset in adulthood and to ...evaluate the role of stromal–epithelial interactions in obesity-associated tumorigenesis. We hypothesized that hepatocyte growth factor (HGF) plays a promoting role in BBC, which express the HGF receptor, c-Met. In C3(1)-T
Ag
mice, a murine model of BBC, we demonstrated that obesity leads to a significant increase in HGF secretion and an associated decrease in tumor latency. By immunohistochemical analysis, normal mammary gland exhibited obesity-induced HGF, c-Met and phospho-c-Met, indicating that the activation of the cascade was obesity-driven. HGF secretion was also increased from primary mammary fibroblasts isolated from normal mammary glands and tumors of obese mice compared to lean. These results demonstrate that obesity-induced elevation of HGF expression is a stable phenotype, maintained after several passages, and after removal of dietary stimulation. Conditioned media from primary tumor fibroblasts from obese mice drove tumor cell proliferation. In co-culture, neutralization of secreted HGF blunted tumor cell migration, further linking obesity-mediated HGF-dependent effects to in vitro measures of tumor aggressiveness. In sum, these results demonstrate that HGF/c-Met plays an important role in obesity-associated carcinogenesis. Understanding the effects of obesity on risk and progression is important given that epidemiologic studies imply a portion of BBC could be eliminated by reducing obesity.
Basal-like and luminal breast cancers have distinct stromal-epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal-epithelial ...interactions evolve in benign and pre-invasive lesions.
To study epithelial-stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis.
Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basallike subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival.
Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.
PDF
