Quanwen Li

Three-Dimensional Overlay Culture Models of Human Breast Cancer Reveal a Critical Sensitivity to Mitogen-Activated Protein Kinase Kinase Inhibitors

Tumor cells that are grown in three-dimensional (3D) cell culture exhibit relative resistance to cytotoxic drugs compared with their response in conventional two-dimensional (2D) culture. We studied the effects of targeted agents and doxorubicin on 2D and 3D cultures of human breast cell lines that represent the progression from normal epithelia (modeled by MCF10A cells) through hyperplastic variants to a dysplastic/carcinoma phenotype (MCF10.DCIS cells), variants transformed by expression of activated Ras, and also a basal-subtype breast carcinoma cell line (MDA-MB-231). The results showed the expected relative resistance to the cytotoxic agent doxorubicin in 3D cultures, with greater resistance in normal and hyperplastic cells than in carcinoma models. However, the response to the targeted inhibitors was more complex. Inhibition of mitogen-activated protein kinase kinase (MEK) by either 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) or 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (CI-1040, PD184352) produced a similar inhibition of the growth of all the MCF10 cell lines in 2D. In 3D culture, the normal and hyperplastic models exhibited some resistance, whereas the carcinoma models became far more sensitive to MEK inhibition. Increased sensitivity to MEK inhibition was also seen in MDA-MB-231 cells grown in 3D compared with 2D. In contrast, inhibition of phosphatidylinositol 3′-kinase activity by wortmannin had no significant effect on the growth of any of the cells in either 2D or 3D. Our conclusion is that 3D culture models may not only model the relative resistance of tumor cells to cytotoxic therapy but also that the 3D approach may better identify the driving oncogenic pathways and critical targeted inhibitors that may be effective treatment approaches.

RNA-Seq of Human Breast Ductal Carcinoma In Situ Models Reveals Aldehyde Dehydrogenase Isoform 5A1 as a Novel Potential Target

Breast ductal carcinoma in situ (DCIS) is being found in great numbers of women due to the widespread use of mammography. To increase knowledge of DCIS, we determined the expression changes that are common among three DCIS models (MCF10.DCIS, SUM102 and SUM225) compared to the MCF10A model of non-tumorigenic mammary epithelial cells in three dimensional (3D) overlay culture with reconstituted basement membrane (rBM). Extracted mRNA was subjected to 76 cycles of deep sequencing (RNA-Seq) using Illumina Genome Analyzer GAIIx. Analysis of RNA-Seq results showed 295 consistently differentially expressed transcripts in the DCIS models. These differentially expressed genes encode proteins that are associated with a number of signaling pathways such as integrin, fibroblast growth factor and TGFβ signaling, show association with cell-cell signaling, cell-cell adhesion and cell proliferation, and have a notable bias toward localization in the extracellular and plasma membrane compartments. RNA-Seq data was validated by quantitative real-time PCR of selected differentially expressed genes. Aldehyde dehydrogenase 5A1 (ALDH5A1) which is an enzyme that is involved in mitochondrial glutamate metabolism, was over-expressed in all three DCIS models at both the mRNA and protein levels. Disulfiram and valproic acid are known to inhibit ALDH5A1 and are safe for chronic use in humans for other disorders. Both of these drugs significantly inhibited net proliferation of the DCIS 3D rBM overlay models, but had minimal effect on MCF10A 3D rBM overlay models. These results suggest that ALDH5A1 may play an important role in DCIS and potentially serve as a novel molecular therapeutic target.

Tyrosyl phosphorylated PAK1 regulates breast cancer cell motility in response to prolactin through filamin A

The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells.

Images of Cleavage: Tumor Proteases in Action

The roles of proteases in cancer are now known to be much broader than simply degradation of extracellular matrices during tumor invasion and metastasis. Furthermore, proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, and endothelial cells) as well as tumor cells are recognized to contribute to proteolytic pathways critical to neoplastic progression. Although increased expression of proteases at the level of transcripts and protein has been observed in many tumors, the functional roles of proteases remain to be determined. Novel techniques for imaging activity of proteases, both in vitro and in vivo, are available as are selective imaging probes and substrates that allow discrimination of the activity of one class of protease from another or one individual protease from another. In this chapter, we describe in vitro models and assays for the functional imaging of proteases and proteolytic pathways. These models and assays can serve as screening platforms for the identification of pathways that are potential therapeutic targets and for further development of technologies and imaging probes for in vivo use. Such uses might include diagnosis and patient follow-up during the course of therapies that alter protease activities, perhaps even providing the crucial data needed to alter the course of treatment and/or the therapies used.

Phosphorylation of rat kidney Na-K pump at Ser938 is required for rapid angiotensin II-dependent stimulation of activity and trafficking in proximal tubule cells

How angiotensin (ANG) II acutely stimulates the Na-K pump in proximal tubules is only partially understood, limiting insight into how ANG II increases blood pressure. First, we tested whether ANG II increases the number of pumps in plasma membranes of native rat proximal tubules under conditions of rapid activation. We found that exposure to 100 pM ANG II for 2 min, which was previously shown to increase affinity of the Na-K pump for Na and stimulate activity threefold, increased the amount of the Na-K pump in plasma membranes of native tubules by 33%. Second, we tested whether previously observed increases in phosphorylation of the Na-K pump at Ser(938) were part of the stimulatory mechanism. These experiments were carried out in opossum kidney cells, cultured proximal tubules stably coexpressing the ANG type 1 (AT1) receptor, and either wild-type or a S938A mutant of rat kidney Na-K pump under conditions found by others to stimulate activity. We found that 10 min of incubation in 10 pM ANG II stimulated activity of wild-type pumps from 2.3 to 3.5 nmol K · mg protein(-1) · min(-1) and increased the amount of the pump in the plasma membrane by 80% but had no effect on cells expressing the S938A mutant. We conclude that acute stimulation of Na-K pump activity in native rat proximal tubules includes increased trafficking to the plasma membrane and that phosphorylation at Ser(938) is part of the mechanism by which ANG II directly stimulates activity and trafficking of the rat kidney Na-K pump in opossum kidney cells.

Abstract B28: Induced expression of Sprouty4 in breast invasive ductal carcinoma cells inhibits ERK MAP kinase and reduces malignant phenotype

Our previous work has identified increased expression of Sprouty4 in 3D models of breast ductal carcinoma in situ (DCIS). Sprouty4 in other systems has been shown to function as a negative regulator of the mitogen activated protein kinase (MAPK/ERK) pathway. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling in DCIS, and that its loss or reduced expression is one mechanism by which triple-negative lesions progress toward invasive ductal carcinoma (IDC). Using immunohistochemistry our labs have found that Sprouty4 was highly expressed in some human premalignant breast tissue samples, and that this expression was reduced in malignant triple-negative samples. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient over-expression of Sprouty4 reduced both MAPK/ERK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments done in IDC cells overexpressing Sprouty4 revealed relocation of E-cadherin back to the cell surface and the restoration of adherens junctions. To determine if these effects were due to changes in MAPK/ERK signaling, IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug restored an epithelial-like phenotype similar to that produced by Sprouty4 over-expression. From these data we conclude that Sprouty4 may act to control MAPK/ERK signaling in a subset of DCIS, thus limiting the progression of these premalignant breast cancers. Further, if in vivo data correspond with our in vitro work, this may argue for the investigation of MEK inhibitors as an adjunct treatment in triple-negative disease, where options are limited. Citation Format: Ryan M. Jackson, Ethan J. Brock, Seema Shah, Mansoureh Sameni, Bonnie F. Sloane, Quanwen Li, Raymond R. Mattingly. Induced expression of Sprouty4 in breast invasive ductal carcinoma cells inhibits ERK MAP kinase and reduces malignant phenotype. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B28.

Abstract 3150: Exploring the role of Rap1Gap in the progression from DCIS to invasive breast carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Understanding the underlying mechanisms that drive malignant progression of breast cancer is important. The purpose of this study is to define the role of Rap1Gap in the progression of ductal carcinoma in-situ (DCIS) to invasive cancer. We employed an in-vitro three-dimensional (3D) overlay model that provides a physiologically relevant microenvironment to study mechanisms of mammary gland development and malignant progression. We previously employed next generation sequencing (NGS) and identified 63 consistently upregulated genes in three DCIS lines (SUM102, SUM225, MCF10.DCIS) compared to non-transformed mammary epithelial cells. To gain insight into common transcriptional elements of co-regulated genes that may be critical to the DCIS phenotype, we have mined our NGS data using Frameworker (Genomatix). The 63 upregulated genes are associated with 244 candidate promoters out of a total of 82,703 promoters in the entire human genome. Our analysis shows that the common framework RXRF-ZF02-ZF02-PLAG-HDBP is only present in the promoters of three genes: RAP1GAP, SPRY4 and PDGFB. All of these genes are upregulated in our DCIS signature, which means that this framework is hugely enriched (336-fold) over what would be expected by chance. Rap1Gap has tumor suppressor properties; its functions, via regulation of Rap1 activity, include regulation of cell adhesion, suppression of cell proliferation and metastasis. In other epithelial cancers, loss of Rap1Gap has been linked to epithelial to mesenchymal transition (EMT) and invasion. To begin study of Rap1Gap in breast cancer progression, we performed immunohistochemistry. We show strong Rap1Gap staining in DCIS with little to no expression in myoepithelial and stromal components. Immunoblotting results show that Rap1Gap levels in MCF10.CA1d cells (a model of invasive carcinoma) are reduced compared to those in MCF10.DCIS cells. Rap1Gap expression is low in additional basal breast cancer cell lines, but high in luminal ER+ /PR+ cell lines. We propose that over-expression of Rap1Gap may serve as a tumor suppressive mechanism in DCIS; subsequent reduction of Rap1Gap may be a switch for EMT and progression to an invasive phenotype. Citation Format: Seema Shah, Kingsley Osuala, Shihong Mao, Quanwen Li, Bonnie Sloane, Stephen Krawetz, Raymond R. Mattingly. Exploring the role of Rap1Gap in the progression from DCIS to invasive breast carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2014-3150