Laser capture microdissection (LCM) offers a rapid and precise method of isolating and removing specified cells from complex tissues for subsequent analysis of their RNA, DNA, or protein content, thereby allowing assessment of the role of the cell type in the normal physiologic or disease process being studied. In this unit, protocols for the preparation of mammalian frozen tissues, fixed tissues, and cytologic specimens for LCM, including hematoxylin and eosin staining, are presented, as well as a protocol for the performance of LCM utilizing the PixCell I or II Laser Capture Microdissection System manufactured by Arcturus Engineering. Also provided is a protocol for tissue processing and paraffin embedding, and recipes for lysis buffers for the recovery of nucleic acids and proteins. The Commentary section addresses the types of specimens that can be utilized for LCM and approaches to staining of specimens for cell visualization. Emphasis is placed on the preparation of tissue or cytologic specimens as this is critical to effective LCM.
CDC6 plays a critical role in regulation of the onset of DNA replication in eukaryotic cells. We have found thatCdc6 expression is down-regulated in prostate cancer as detected by semiquantitative reverse transcriptase-PCR of prostate cell lines and laser-captured microdissected prostate tissues. This result was substantiated by immunohistochemical analysis of paraffin-embedded tissue sections and immunoblot analysis of benign (BPH-1) and adenocarcinomatous prostatic cells. Furthermore, a 100-fold reduction in the transcription efficiency of the Cdc6promoter-luciferase construct was noted in the metastatic PC3 cells compared with that in BPH-1 cells. Concentration of the E2F and Oct1 transcription factors that have putative binding sites in theCdc6 promoter was substantially low in PC3 cells compared with BPH cells. Mutagenesis of the two E2F binding sites on theCdc6 promoter resulted in increased promoter activity in PC3 cells owing to elimination of the negative regulation by pRb·E2F complex but not to the level of that obtained in BPH cells. We conclude that an altered interaction of transcription factors may be responsible for the down-regulation of Cdc6transcription in PC3 cells. Our study suggests a potential use of the lack of CDC6 expression as an index of prostate cancer development.
Mammalian LIM kinase 1 (LIMK1) is involved in reorganization of actin cytoskeleton through inactivating phosphorylation of the ADF family protein cofilin, which depolymerizes actin filaments. Maintenance of the actin dynamics in an ordered fashion is essential for stabilization of cell shape or promotion of cell motility depending on the cell type. These are the two key phenomena that may become altered during acquisition of the metastatic phenotype by cancer cells. Here we show that LIMK1 is overexpressed in prostate tumors and in prostate cancer cell lines, that the concentration of phosphorylated cofilin is higher in metastatic prostate cancer cells, and that a partial reduction of LIMK1 altered cell proliferation by arresting cells at G2/M, changed cell shape, and abolished the invasiveness of metastatic prostate cancer cells. We also show that the ectopic expression of LIMK1 promotes acquisition of invasive phenotype by the benign prostate epithelial cells. Our data provide evidence of a novel role of LIMK1 in regulating cell division and invasive property of prostate cancer cells and indicate that the effect is not mediated by phosphorylation of cofilin. Our study correlates with the recent observations showing a metastasis-associated chromosomal gain on 7q11.2 in prostate cancer, suggesting a possible gain in LIMK1 DNA (7q11.23).
Wnt5a is a non-canonical signaling Wnt that has been implicated in tumor suppression. We previously showed that loss of Wnt5a in MMTV-PyVmT tumors resulted in a switch in tumor phenotype resulting in tumors with increased basal phenotype and high Wnt/β-catenin signaling. The object of this study was to test the hypothesis that Wnt5a can act to inhibit tumors formed by activation of Wnt/β-catenin signaling. To this end, we characterized tumor and non-tumor mammary tissue from MMTV-Wnt1 and double transgenic MMTV-Wnt1;MMTV-Wnt5a mice. Wnt5a containing mice demonstrated fewer tumors with increased latency when compared to MMTV-Wnt1 controls. Expression of markers for basal-like tumors was down-regulated in the tumors that formed in the presence of Wnt5a indicating a phenotypic switch. Reduced canonical Wnt signaling was detected in double transgenic tumors as a decrease in active β-catenin protein and a decrease in Axin2 mRNA transcript levels. In non-tumor tissues, over-expression of Wnt5a in MMTV-Wnt1 mammary glands resulted in attenuation of phenotypes normally observed in MMTV-Wnt1 glands including hyperbranching and increased progenitor and basal cell populations. Even though Wnt5a could antagonize Wnt/β-catenin signaling in primary mammary epithelial cells in culture, reduced Wnt/β-catenin signaling was not detected in non-tumor MMTV-Wnt1;Wnt5a tissue in vivo. The data demonstrate that Wnt5a suppresses tumor formation and promotes a phenotypic shift in MMTV-Wnt1 tumors.
Abstract 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 < 0.001) and migration (222%; p < 0.05) compared to untreated cells, whereas full length adiponectin had no significant effect. Rapamycin, an established autophagy inducer, elicited effects similar to globular adiponectin (increase of 210%; p < 0.001 in invasion and 238%; p < 0.05 in migration). Likewise, three-dimensional growth in Matrigel revealed that cells treated with globular adiponectin and rapamycin developed extended spikes indicative of a more invasive phenotype, whereas those treated with full-length adiponectin maintained a less invasive grape-like structure. Neither adiponectin isoform altered proliferation. Biochemical assays of autophagic induction supported these observations, demonstrating increases in LC3B-II expression (immunoblot) and in the number of intracellular LC3B puncta (immunofluorescence) upon treatment with globular, but not full-length, adiponectin. Together, these results suggest a plausible model linking a specific adiponectin isoform with induction of autophagy to stimulate breast cancer metastasis. Our findings will advance the field by revealing distinct, novel roles for key microenvironmental regulatory molecules, potentially opening up new avenues for therapeutic development. This work is timely considering recent interest in adiponectin and adiponectin receptor agonists as therapeutic strategies. In addition, noting that an aberrant level of adiponectin is a putative mechanism linking obesity to poor breast cancer prognosis and metastasis, our results may provide mechanistic insight to guide lifestyle interventions that will reduce breast cancer's heavy morbidity and mortality burden. 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
