Emily Falk Libby

A ferret model of COPD-related chronic bronchitis

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US. The majority of COPD patients have symptoms of chronic bronchitis, which lacks specific therapies. A major impediment to therapeutic development has been the absence of animal models that recapitulate key clinical and pathologic features of human disease. Ferrets are well suited for the investigation of the significance of respiratory diseases, given prior data indicating similarities to human airway physiology and submucosal gland distribution. Here, we exposed ferrets to chronic cigarette smoke and found them to approximate complex clinical features of human COPD. Unlike mice, which develop solely emphysema, smoke-exposed ferrets exhibited markedly higher numbers of early-morning spontaneous coughs and sporadic infectious exacerbations as well as a higher level of airway obstruction accompanied by goblet cell metaplasia/hyperplasia and increased mucus expression in small airways, indicative of chronic bronchitis and bronchiolitis. Overall, we demonstrate the first COPD animal model exhibiting clinical and pathologic features of chronic bronchitis to our knowledge, providing a key advance that will greatly facilitate the preclinical development of novel treatments for this disease.

Globular adiponectin enhances invasion in human breast cancer cells

Every year, a large number of women succumb to metastatic breast cancer due to a lack of curative approaches for this disease. Adiponectin (AdipoQ) is the most abundant of the adipocyte-secreted adipokines. In recent years, there has been an interest in the use of AdipoQ and AdipoQ receptor agonists as therapeutic agents for the treatment of breast cancer. However, while multiple epidemiological studies have previously indicated that low levels of circulating plasma AdipoQ portend poor prognosis in patients with breast cancer, recent studies have reported that elevated expression levels of AdipoQ in breast tissue are correlated with advanced stages of the disease. Thus, the aim of the present study was to clarify the mechanism by which AdipoQ in breast tissue acts directly on tumor cells to regulate the early steps of breast cancer metastasis. In the present study, the effects of different AdipoQ isoforms on the metastatic potential of human breast cancer cells were investigated. The results revealed that globular adiponectin (gAd) promoted invasive cell morphology and significantly increased the migration and invasion abilities of breast cancer cells, whereas full-length adiponectin (fAd) had no effect on these cells. Additionally, gAd, but not fAd, increased the expression levels of microtubule-associated protein 1 light chain 3 beta (LC3B)-II and intracellular LC3B puncta, which are indicators of autophagosome formation, thus suggesting autophagic induction by gAd. Furthermore, the inhibition of autophagic function by autophagy-related protein 7 knockdown attenuated the gAd-induced increase in invasiveness in breast cancer cells. Therefore, the results of the present study suggested that a specific AdipoQ isoform may enhance breast cancer invasion, possibly via autophagic induction. Understanding the roles of the different AdipoQ isoforms as microenvironmental regulatory molecules may aid the development of effective AdipoQ-based treatments for breast cancer.

Linking adiponectin and autophagy in the regulation of breast cancer metastasis

Adipokines within the tumor microenvironment may play important roles in regulating the early steps of breast cancer metastasis. Adiponectin (AdipoQ) is the most abundant adipokine and exists in multiple forms: full-length multimers (fAd) and a cleaved, globular isoform (gAd). While these isoforms are observed as having distinct biological properties, nearly all investigation into AdipoQ in breast cancer has focused on the antitumor roles of fAd, while mostly ignoring gAd. However, evidence from other disease settings suggests that gAd is linked to processes known to promote metastasis. Here, we discuss key areas in which knowledge about AdipoQ in breast cancer is lacking, expressly focusing on data suggesting that gAd is elevated in the microenvironment and may act directly on invasive breast cancer cells to support their initial metastatic progression. We discuss autophagy as a potential mechanism of action for this effect. Overall, given that AdipoQ and AdipoQ receptor agonists have been proposed as therapeutic strategies, it is necessary to better understand the various functions of these regulatory molecules in metastatic breast cancer. Doing so will help ensure the most effective approaches to treating this disease, for which there remain no curative options.

Therapeutic benefit observed with the CFTR potentiator, ivacaftor, in a CF patient homozygous for the W1282X CFTR nonsense mutation

Premature termination codons (PTCs) in cystic fibrosis transmembrane conductance regulator (CFTR) gene result in nonfunctional CFTR protein and are the proximate cause of ~11% of CF causing alleles. Aminoglycosides and other novel agents are known to induce translational readthrough of PTCs, a potential therapeutic approach. Among PTCs, W1282X CFTR is unique, as it is a C-terminal CFTR mutation that can exhibit partial activity, even in the truncated state. The potentiator ivacaftor (VX-770) is approved for treating CF patients with G551D and other gating mutations. Based on previous studies demonstrating the beneficial effect of ivacaftor for PTC mutations following readthrough in vitro, we hypothesized that ivacaftor may enhance CFTR activity in CF patients expressing W1282X CFTR, and could be further enhanced by readthrough. Ivacaftor significantly increased CFTR activity in W1282X-expressing cells compared to R1162X CFTR cells, and was further enhanced by readthrough with the aminoglycoside G418. Primary nasal epithelial cells from a W1282X homozygous patient showed improved CFTR function in the presence of ivacaftor. Upon ivacaftor administration to the same patient, there was significant improvement in pulmonary exacerbation frequency, BMI, and insulin requirement, whereas FEV1 remained stable over 3years. These studies suggest that ivacaftor may have moderate clinical benefit in patients with preserved expression of the W1282X CFTR mutation by stimulating residual activity of the truncated protein, suggesting the need for further studies including the addition of efficacious readthrough agents.

SIN3A and SIN3B differentially regulate breast cancer metastasis

SIN3 corepressor complexes play important roles in both normal development and breast cancer. Mammalian cells have two paralogs of SIN3 (SIN3A and SIN3B) that are encoded by distinct genes and have unique functions in many developmental processes. However, specific roles for SIN3A and SIN3B in breast cancer progression have not been characterized. We generated stable knockdown cells of SIN3 paralogs individually and in combination using three non-overlapping shRNA. Stable knockdown of SIN3B caused a significant decrease in transwell invasion through Matrigel and decreased the number of invasive colonies when grown in a 3D extracellular matrix. Conversely, stable knockdown of SIN3A significantly increased transwell invasion and increased the number of invasive colonies. These results were corroborated in vivo in which SIN3B knockdown significantly decreased and SIN3A knockdown increased experimental lung metastases. RNA sequencing was used to identify unique targets and biological pathways that were altered upon knockdown of SIN3A compared to SIN3B. Additionally, we analyzed microarray data sets to identify correlations of SIN3A and SIN3B expression with survival in patients with breast cancer. These data sets indicated that high mRNA expression of SIN3A as well as low mRNA expression of SIN3B correlates with longer relapse free survival specifically in patients with triple negative breast cancer which corresponds with our in vitro and in vivo data. These results demonstrate key functional differences between SIN3 paralogs in regulating the process of breast cancer metastasis and suggest metastasis suppressive roles of SIN3A and metastasis promoting roles of SIN3B.

Obesity is associated with higher 4E-BP1 expression in endometrial cancer

Purpose Obesity is associated with risk and prognosis of endometrial cancer (EC), and the mammalian target of rapamycin complex 1 (mTORC1) pathway may play an instrumental role. We sought to explore the associations between cellular proliferation, Akt, and 4E binding protein-1 (4E-BP1) (a downstream target of mTORC1), in obese and nonobese women with and without EC. Methods Archival tissue-specimens from endometrial biopsies were grouped into two broad categories based on the observed disease behavior and similarities in tissue staining patterns: benign/hyperplasia (without cytologic atypia) (n=18) versus atypia (complex hyperplasia with cytologic atypia)/carcinoma (n=25). The characteristics of the study population, including height and weight to determine body mass index (BMI: kg/m2), were abstracted from medical records. Immunohistochemistry was used to assess the phosphorylated (p)Akt, p4E-BP1, and antigen Ki67. Results Cytoplasmic and nuclear pAkt were significantly associated with cytoplasmic p4E-BP1 (ρ=+0.48, ρ=+0.50) (P<0.05) and nuclear p4E-BP1 (ρ=+0.40, ρ=+0.44) (P<0.05); cytoplasmic and nuclear p4E-BP1 were significantly associated with Ki67 (ρ=+0.46, ρ=+0.59) (P<0.05). Compared with the benign/hyperplasia group, the women with atypia/carcinoma had significantly higher cytoplasmic and nuclear p4E-BP1 and Ki67. This staining pattern was similar in obese women; however, in nonobese women, neither cytoplasmic nor nuclear p4E-BP1staining differed between benign/hyperplasia versus atypia/carcinoma. Conclusion The activation of 4E-BP1 was higher in the obese women with EC. Adiposity may be a key factor to consider in future studies investigating the role of 4E-BP1 as a biomarker and therapeutic target in EC.

Increased autophagic response in a population of metastatic breast cancer cells

Breast cancer cells are heterogeneous in their ability to invade and fully metastasize, and thus also in their capacity to survive the numerous stresses encountered throughout the multiple steps of the metastatic cascade. Considering the role of autophagy as a survival response to stress, the present study hypothesized that distinct populations of breast cancer cells may possess an altered autophagic capacity that influences their metastatic potential. It was observed that a metastatic breast cancer cell line, MDA-MB-231, that was sensitive to autophagic induction additionally possessed the ability to proliferate following nutrient deprivation. Furthermore, a selected subpopulation of these cells that survived multiple exposures to starvation conditions demonstrated a heightened response to autophagic induction compared to their parent cells. Although this subpopulation maintained a more grape-like pattern in three-dimensional culture compared to the extended spikes of the parent population, autophagic induction in this subpopulation elicited an invasive phenotype with extended spikes. Taken together, these results suggest that autophagic induction may contribute to the ability of distinct breast cancer cell populations to survive and invade.

Abstract 989: Regulation of metastatic potential through adiponectin-stimulated induction of autophagy

Each year, an unacceptable number of deaths occur in women because of the lack of curative approaches for metastatic breast cancer. Some of the key regulators of breast cancer progression are molecules within the tumor microenvironment including adipokines, a broad array of autocrine-, endocrine-, and paracrine-acting bioactive molecules secreted by adipocytes. Adiponectin is one of the most abundant adipokines and, while multiple widely cited epidemiological studies have indicated that low levels of circulating plasma adiponectin portend poorer prognosis, recent work has reported that elevated adiponectin expression in breast tissue is, in fact, correlated with more advanced disease. Thus, the purpose of this work is to better understand how adiponectin in breast tissue acts directly on tumor cells to regulate the early steps of breast cancer metastasis. Our hypothesis is that adiponectin alters metastatic potential of breast cancer cells via induction of autophagy. To begin to test this premise, we discerned the effect of globular versus full-length adiponectin on invasive and migratory phenotypes of a human metastatic breast cancer cell line (MDA-MB-231). In transwell assays with and without Matrigel, globular adiponectin increased invasion (91%; p Citation Format: Emily Falk Libby, Jianzhong Liu, Monica J. Lewis, Andra R. Frost, Wendy Demark-Wahnefried, Douglas R. Hurst. Regulation of metastatic potential through adiponectin-stimulated induction of autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 989. doi:10.1158/1538-7445.AM2015-989

No and low alcohol intake may have differential effects on risk of overall and cause-specific mortality

Dear Sir: We read with great interest the article by Vergnaud et al (1) on the relation between adherence to the World Cancer Research Fund (WCRF)/American Institute for Cancer Research (AICR) guidelines and risk of death in Europe. This well-crafted, large-scale study conducted in participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort offers valuable data regarding the impact of the WCRF/AICR recommendations on reducing total and cause-specific mortality and suggests that the utility of these guidelines may extend beyond the scope of cancer prevention. We are, however, keen on gaining additional understanding of the results presented in their Table 4: namely, the risk of death associated with alcohol consumption. The authors found that adherence to the WCRF/AICR recommendation for daily alcohol intake (≤2 drinks for men and 1 drink for women) was protective against all-cause mortality in men but not in women. This result was based on a scoring system that operationalized this alcohol-specific guideline into 3 categories of ethanol intake: ≤20, >20 to ≤30, and >30 g/d for men and ≤10, >10 to ≤20, and >20 g/d for women. Among the 257,421 male study participants, the men whose ethanol intake was >20 to ≤30 g/d had a significantly reduced risk of death compared with men whose consumption exceeded 30 g/d (HR: 0.80), as did men who limited their intake to ≤20 g/d compared with the same referent (HR: 0.89). However, significant associations between risk of death and the alcoholic drinks component of the WCRF/AICR recommendations were not observed among the 121,443 female study participants. We are highly curious both to learn whether making the distinction between no and low ethanol intake would alter the results of this analysis and to see the stratification of HRs by cause of death. Whereas it is widely acknowledged that, unlike in cardiovascular disease, the lowest alcohol-related cancer risk is in fact conferred in the absence of alcohol consumption (2), there remains uncertainty regarding whether the protective effect of abstinence on cancer risk translates to survival outcomes. The most current estimate of alcohol-attributable cancer mortality in the United States to our knowledge suggests that alcohol consumption at any level not only increases cancer risk but, more critically, is a major factor behind cancer-related death in men and women (3). Interestingly, the number of alcohol-attributable deaths was highest for female breast cancer in this investigation. A meta-analysis by Bagnardi et al (4) that included 222 articles concerning alcohol consumption and cancer found that light alcohol drinking (≤1 drink/d) was associated with breast cancer death. In contrast and illustrative of the ambiguity related to drinking and cancer mortality, another recent study reported that any alcohol consumption either before or after breast cancer diagnosis had no adverse impact on survival from breast cancer, cardiovascular disease, or other cause, and that moderate consumption may even have a survival benefit (5). The robust data set of Vergnaud et al presents an opportunity for additional analyses that could shed further light on the advantages or lack thereof of teetotaling in the prevention of cancer or other chronic diseases. As such, we appreciate the authors’ consideration of our request that they both reoperationalize the alcohol-specific WCRF/AICR score such that 0 g/d of ethanol intake is assigned its own category and evaluate alcohol-specific mortality by cause of death and share these results.

Abstract 151: Obesity is independently associated with mTORC1 pathway activation in the progression of endometrial cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Obesity is a major risk factor for endometrial cancer (EC) yet, with the exception of estrogen, the mechanisms underlying this association remain unclear. The mammalian target of rapamycin complex 1 (mTORC1) cascade links energy balance to cell proliferation via the PI3K/Akt pathway. Phosphorylation of Akt by hormone and growth factor signaling activates mTORC1 and results in the downstream phosphorylation of 4EBP1. Activated 4EBP1 positively regulates cap-dependent translation and cell growth, and correlates with EC progression. Thus, we explored the associations between obesity and pAkt and p4EBP1 expression, and cell proliferation rates (Ki67) in benign and malignant endometrial tissues. Methods: Medical records were reviewed to identify women who underwent endometrial biopsy at UAB Hospital from 2008-2010. Women who had recurrent EC or diagnoses of any other cancer were excluded. Data on diagnosis, height, weight, race, age, menopausal status, number of children, medications, and comorbid conditions were extracted from medical records. Height and weight were used to calculate body mass index (BMI: kg/m2). A gynecologic pathologist reviewed H&E slides for concordance with the pathology report, and additional sections were obtained from formalin-fixed paraffin-embedded tissue blocks. Immunohistochemistry was used to assess p4EBP1, pAkt, and Ki67. The pathologist employed a semiquantitative scoring system based on percent positive and staining intensity for p4EBP1 and pAkt, and scored on percent positive for Ki67. Wilcoxon rank-sum tests were used to compare marker activity between women with and without EC in the overall and BMI-stratified sample. Results: Immunohistochemical data were available for 18 benign and 25 type 1 endometrioid EC cases, of which 29 were obese (BMI > 29.9) and 14 were non-obese (BMI ≤ 29.9); the overall mean BMI was 35.5 (SD=11.6). Mean age was 56.7 (12.2). Women with vs. without EC had significantly elevated median [interquartile range] expression of cytoplasmic p4EBP1 (95 [30-180] vs. 0 [0-20], p=0.001), nuclear p4EBP1 (170 [95-195] vs. 70 [20-100], p=0.003), and Ki67 (50 [40-80] vs. 10 [5-20], p<0.0001). EC was not associated with pAKT activity. While obese women with vs. without EC had significantly higher expression of cytoplasmic p4EBP1 (100 [80-180] vs. 0 [0-10], p=0.002) and nuclear p4EBP1 (180 [100-200] vs. 65 [20-100], p=0.002), neither cytoplasmic nor nuclear p4EBP1 activity differed by EC status among non-obese women. No associations between obesity and Ki67 or pAKT were detected. Conclusions: Results from this exploratory study suggest that p4EBP1 expression is elevated in obese women with EC. These preliminary findings suggest that obesity may play a role in EC-related activation of the mTORC1 pathway and 4EBP1, a biomarker for EC outcomes. Citation Format: Emily Falk Libby, Maria Azrad, Lea Novak, Wendy Demark-Wahnefried. Obesity is independently associated with mTORC1 pathway activation in the progression of endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 151. doi:10.1158/1538-7445.AM2013-151