Ryan H. Livengood

KAP1 Promotes Proliferation and Metastatic Progression of Breast Cancer Cells

KAP1 (TRIM28) is a transcriptional regulator in embryonic development that controls stem cell self-renewal, chromatin organization, and the DNA damage response, acting as an essential corepressor for KRAB family zinc finger proteins (KRAB-ZNF). To gain insight into the function of this large gene family, we developed an antibody that recognizes the conserved zinc fingers linker region (ZnFL) in multiple KRAB-ZNF. Here, we report that the expression of many KRAB-ZNF along with active SUMOlyated KAP1 is elevated widely in human breast cancers. KAP1 silencing in breast cancer cells reduced proliferation and inhibited the growth and metastasis of tumor xenografts. Conversely, KAP1 overexpression stimulated cell proliferation and tumor growth. In cells where KAP1 was silenced, we identified multiple downregulated genes linked to tumor progression and metastasis, including EREG/epiregulin, PTGS2/COX2, MMP1, MMP2, and CD44, along with downregulation of multiple KRAB-ZNF proteins. KAP1-dependent stabilization of KRAB-ZNF required direct interactions with KAP1. Together, our results show that KAP1-mediated stimulation of multiple KRAB-ZNF contributes to the growth and metastasis of breast cancer.

NEDD9 Depletion Destabilizes Aurora A Kinase and Heightens the Efficacy of Aurora A Inhibitors: Implications for Treatment of Metastatic Solid Tumors

Aurora A kinase (AURKA) is overexpressed in 96% of human cancers and is considered an independent marker of poor prognosis. While the majority of tumors have elevated levels of AURKA protein, few have AURKA gene amplification, implying that posttranscriptional mechanisms regulating AURKA protein levels are significant. Here, we show that NEDD9, a known activator of AURKA, is directly involved in AURKA stability. Analysis of a comprehensive breast cancer tissue microarray revealed a tight correlation between the expression of both proteins, significantly corresponding with increased prognostic value. A decrease in AURKA, concomitant with increased ubiquitination and proteasome-dependent degradation, occurs due to depletion or knockout of NEDD9. Reexpression of wild-type NEDD9 was sufficient to rescue the observed phenomenon. Binding of NEDD9 to AURKA is critical for AURKA stabilization, as mutation of S296E was sufficient to disrupt binding and led to reduced AURKA protein levels. NEDD9 confers AURKA stability by limiting the binding of the cdh1-substrate recognition subunit of APC/C ubiquitin ligase to AURKA. Depletion of NEDD9 in tumor cells increases sensitivity to AURKA inhibitors. Combination therapy with NEDD9 short hairpin RNAs and AURKA inhibitors impairs tumor growth and distant metastasis in mice harboring xenografts of breast tumors. Collectively, our findings provide rationale for the use of AURKA inhibitors in treatment of metastatic tumors and predict the sensitivity of the patients to AURKA inhibitors based on NEDD9 expression.

NEDD9 Regulates Actin Dynamics through Cortactin Deacetylation in an AURKA/HDAC6–Dependent Manner

The prometastatic protein NEDD9 (neural precursor cell expressed, developmentally downregulated 9) is highly expressed in many cancers and is required for mesenchymal individual cell migration and progression to the invasive stage. Nevertheless, the molecular mechanisms of NEDD9-driven migration and the downstream targets effecting metastasis are not well defined. In the current study, knockdown of NEDD9 in highly metastatic tumor cells drastically reduces their migratory capacity due to disruption of actin dynamics at the leading edge. Specifically, NEDD9 deficiency leads to a decrease in the persistence and stability of lamellipodial protrusions similar to knockdown of cortactin (CTTN). Mechanistically, it was shown that NEDD9 binds to and regulates acetylation of CTTN in an Aurora A kinase (AURKA)/HDAC6–dependent manner. The knockdown of NEDD9 or AURKA results in an increase in the amount of acetylated CTTN and a decrease in the binding of CTTN to F-actin. Overexpression of the deacetylation mimicking (9KR) mutant of CTTN is sufficient to restore actin dynamics at the leading edge and migration proficiency of the tumor cells. Inhibition of AURKA and HDAC6 activity by alisertib and Tubastatin A in xenograft models of breast cancer leads to a decrease in the number of pulmonary metastases. Collectively, these findings identify CTTN as the key downstream component of NEDD9-driven migration and metastatic phenotypes. Implications: This study provides a mechanistic platform for therapeutic interventions based on AURKA and HDAC6 inhibition for patients with metastatic breast cancer to prevent and/or eradicate metastases. Mol Cancer Res; 12(5); 681–93. ©2014 AACR.

NEDD9 Depletion Leads to MMP14 Inactivation by TIMP2 and Prevents Invasion and Metastasis.

The scaffolding protein NEDD9 is an established prometastatic marker in several cancers. Nevertheless, the molecular mechanisms of NEDD9-driven metastasis in cancers remain ill-defined. Here, using a comprehensive breast cancer tissue microarray, it was shown that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly, it was shown that NEDD9 overexpression is a hallmark of highly invasive breast cancer cells. Moreover, NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo, leading to decreased circulating tumor cells and lung metastases in xenograft models. Mechanistically, NEDD9 localized to invasive pseudopods and was required for local matrix degradation. Depletion of NEDD9 impaired invasion of cancer cells through inactivation of membrane-bound matrix metalloproteinase MMP14 by excess TIMP2 on the cell surface. Inactivation of MMP14 is accompanied by reduced collagenolytic activity of soluble metalloproteinases MMP2 and MMP9. Reexpression of NEDD9 is sufficient to restore the activity of MMP14 and the invasive properties of breast cancer cells in vitro and in vivo. Collectively, these findings uncover critical steps in NEDD9-dependent invasion of breast cancer cells. Implications: This study provides a mechanistic basis for potential therapeutic interventions to prevent metastasis. Mol Cancer Res; 12(1); 69–81. ©2013 AACR.

Combination of Eribulin and Aurora A Inhibitor MLN8237 Prevents Metastatic Colonization and Induces Cytotoxic Autophagy in Breast Cancer

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule-targeting drug eribulin on different stages of metastasis in triple-negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process, including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells, providing foundation for the combination therapy. Mechanistically, AURKA inhibition induces cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. These preclinical data provide a new understanding of the mechanisms by which MLN8237 mediates its antimetastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early-stage solid tumors. Mol Cancer Ther; 15(8); 1809–22. ©2016 AACR.

Dietary n-3 polyunsaturated fatty acids or soy protein isolate did not attenuate disease progression in a female rat model of autosomal recessive polycystic kidney disease

Polycystic kidney disease (PKD) is an incurable genetic disorder that is characterized by multiple benign cysts. As PKD advances, cyst growth increases kidney volume, decreases renal function, and may lead to end-stage renal disease; however, in a PKD rat model, feeding soy protein isolate (SPI) reduced cyst proliferation and growth. The n-3 polyunsaturated fatty acids (PUFAs) are noted for their anti-inflammatory actions. Therefore, diet therapy could offer a potentially efficacious, safe, and cost-effective strategy for treating PKD. The objective of this study was to investigate the role of soy protein and/or n-3 PUFAs on PKD progression and severity in the rat model of autosomal recessive PKD. We hypothesized that the antiproliferative and anti-inflammatory actions associated with soy protein and n-3 PUFA supplementation will attenuate PKD progression in female PCK rats. For 12 weeks, young (age, 28 days) female PCK rats were randomly assigned (n=12/group) to 4 different diets: casein±corn oil, casein±soybean oil, SPI±soybean oil, or SPI±1:1 soybean/salmon oil (SPI±SB). The feeding of the different protein and lipid sources had no significant effect on relative kidney weight. Histologic evaluation showed no significant differences in cortical or medullary cyst size, interstitial inflammation, and fibrosis among diet groups. However, rats fed SPI±SB diet had cortical cyst obstruction and the highest (P<.01) serum blood urea nitrogen concentration. Rats fed SPI±SB diet had the highest (P<.001) renal docosahexaeonic acid, but there were no significant differences in renal tissue inflammation and proliferation gene expression among the diet groups. Based on these results, dietary soy protein and/or n-3 PUFAs did not attenuate disease progression or severity in the female PCK rat model of autosomal recessive PKD.

Hyperthermic tissue sealing devices: a proposed histopathologic protocol for standardizing the evaluation of thermally sealed vessels

Hyperthermic tissue sealing devices are advancing modern laparoscopy and other minimally invasive surgical approaches. Histopathologic evaluation of thermally sealed vessels can provide important information on their associated tissue effects and reactions. However, a standardized systematic approach has not been historically used in the literature. This paper proposes a histologic approach for the analysis of thermally sealed vessels and their basis of hemostasis, including thermal tissue changes, healing, and thrombosis. Histologic evaluation during the first week (Days 3-7) can assess the seals primary tissue properties. These parameters include the thermal seals length, architecture, tissue layers involved, adventitial collagen denaturation length, entrapped vapor or blood pockets, tissue homogenization and thermal tissue injury zones. While the architectural features can be assessed in Day 0-3 specimens, the latter thermal injury zones are essentially not assessable in Day 0-3 specimens. Day 14 specimens can provide information on the early healing response to the sealed vessel. Day 30 and longer specimens can be used to evaluate the seals healing reactions. Assessment of the healing response should include seal site inflammation, granulation tissue, necrosis resorption, fibroproliferative scar healing, and thrombus organization. In order to accurately evaluate these parameters, careful specimen orientation, embedding and multiple histologic sections across the entire seal width are required. When appropriate in vivo post-treatment times are used, thermal vessel seals can be evaluated with routine light microscopy and common histologic staining methods.

Non-ablative hyperthermic mesenchymal regeneration: a proposed mechanism of action based on the Vivev model

Novel non-ablative hyperthermic medical devices are currently being developed, in association with cryogen surface cooling, to rejuvenate tissues without collagen scarring. These devices have been designed to remodel skin, manage urinary stress incontinence, and more recently, treat vaginal laxity. In contrast to the thermal injury and reparative healing associated with higher energy ablation systems, these lower energy non-ablative systems are designed to subtly modify the collagen, stimulate the fibroblasts, and maintain a functional tissue architecture that subsequently promotes tissue rejuvenation and restoration. While these devices have primarily relied on clinical outcome questionnaires and satisfaction surveys to establish efficacy, a physiologic explanation for the induced tissue changes and tightening has not been well documented. Recent histology studies, using the Viveve ovine vaginal treatment model, have identified changes that propose both a mechanism of action and a tissue remodeling timeline for such non-ablative hyperthermic devices. The Viveve model results are consistent with subtle connective tissue changes leading to fibroblast stimulation and subsequent collagen replacement and augmentation. Unlike tissue ablation devices that cause thermal necrosis, these non-ablative devices renew the targeted tissue without dense collagenous scarring over a period of 3 or more months. The spectrum of histologic findings, as illustrated in the Viveve ovine vaginal model, further support the previously documented safety and efficacy profiles for low-dose non-ablative hyperthermic devices that rejuvenate and tighten submucosal tissues.

Feeding soy protein isolate and n-3 PUFA affects polycystic liver disease progression in a PCK rat model of autosomal polycystic kidney disease.

Objective: In polycystic liver disease (PCLD), multiple cysts cause liver enlargement, structural damage, and loss of function. Soy protein and dietary &ohgr;-3 polyunsaturated fatty acids (n-3 PUFAs) have been found to decrease cyst proliferation and inflammation in polycystic kidney disease. Therefore, the aim of the study was to investigate whether soy protein and n-3 PUFA supplementation attenuates PCLD. Methods: Young (age 28 days) female PCK rats were fed (n = 12 per group) either casein + corn oil (casein + CO), casein + soybean oil (casein + SO), soy protein isolate + soybean oil (SPI + SO), or SPI + 1:1 soybean/salmon oil blend (SPI + SB) diet for 12 weeks. Liver histology, gene expression by real-time quantitative polymerase chain reaction, and serum markers of liver injury were determined. Results: Diet had no effect on PCLD progression as indicated by no significant differences in liver weight and hepatic proliferation gene expression between diet groups. PCK rats fed SPI + SB diet, however, had the greatest (P < 0.05) histological evidence of hepatic cyst obstruction, portal inflammation, steatosis, and upregulation (P = 0.03) of fibrosis-related genes. Rats fed SPI + SB diet also had the lowest (P < 0.001) serum cholesterol and higher (P < 0.05) serum alkaline phosphatase and bilirubin concentrations. Conclusions: Feeding young female PCK rats SPI and n-3 PUFA failed to attenuate PCLD progression. Furthermore, feeding SPI + SB diet resulted in complications of hepatic steatosis attributable to cysts obstruction of bile duct and hepatic vein. Based on the results, it was concluded that diet intervention alone was not effective at attenuating PCLD associated with autosomal recessive polycystic kidney disease.

Two-Dimensional Echocardiographic Findings of Pulmonary Valve Cyst Secondary to Pulmonary Valvuloplasty

Real time two‐dimensional transthoracic and transesophageal echocardiography demonstrated a mobile echolucent mass attached to the pulmonary valve in a 25‐year‐old adult, 20 years following balloon pulmonary valvuloplasty. The mass was surgically excised and pathology showed it to be a cyst.