The Effects of Tks5 Alternative Splicing on Invadopodia Activity in Prostate Cancer Cells

G. Travis Tabor, Joey H. Breeyear, Matthew C. Sanderson, Darren F. Seals

Department of Biology, Appalachian State University, Boone, NC 28608 Actin ECM Invadopodia

qRT-PCR Quality Control Each primer set was tested on all hTks5 splice form constructs and the products were analyzed via gel electrophoresis. The annealing temperature was 51°C and 30 PCR cycles were performed.

1.  The three longer Tks5 splice forms induced significantly more gelatin degradation than the short form when exogenously expressed in LNCaP cells.

2.  A qRT-PCR strategy for determining the Tks5 splice form

expression profile of human carcinoma cell lines has been developed and is currently being assessed for its validity.

3.  If Tks5 alternative splicing is found to be a tissue-specific

phenomenon indicative of tumor malignancy, the aforementioned qRT-PCR strategy may prove to be a novel diagnostic tool.

Conclusions

Special thanks to Appalachian State University for funding this project, the Office of Student Research for providing an Undergraduate Research Assistantship, and the Honors College for the Partnership Board Research Fund award. Nitric acid was generously donated by Dr. Shea Tuberty of the Department of Biology. In addition, Dr. Ece Karatan provided restriction enzymes and buffers used during the cloning process. We thank Dr. Clare Abram of the University of California, San Francisco for generating some of the original Tks5 constructs used in this study.

Acknowledgements

1. Cancer Cell 2005, 7(2), 155-165. 2. Prostate 2014, 74(2), 134-148. 3. J. Biol. Chem. 2003, 278(19), 16844-16851. 4. J. Cell Sci. 2009, 122(15), 2727-2740. 5. J. Cell Biol. 2008, 182(1), 157-169. 6. J. Vis. Exp. 2012, 66, e4119. 7. Methods Mol. Biol. 2011, 769, 111-136.

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Current Work

Figure 9. Preliminary RT-PCR Analysis of Tks5 Splicing in LNCaP Cells. After 35 cycles, LNCaP cells were observed to express only Tks5-A when the products were visualized in a 2% agarose gel containing 5E(-5)% ethidium bromide. GAPDH (G) was used as the positive control. qRT-PCR Protocol

Absolute quantification of Tks5 splice form mRNA will performed via the SYBR Green method using a Applied Biosystems 7300 Real-Time PCR System. Standard curves containing 3, 30, 300, 3E3, 3E4, and, 3E5 copies of each splice form plasmid will be used to quantify the corresponding cDNA target. All standards and samples will be run in duplicate.

Figure 8. Analysis of qRT-PCR Primer Specificity. Left: Only the hTks5-B primer set was shown to amplify the intended target. The positive control (+) consisted of a primer set targeting a constitutive exon downstream of the splice sites. Right: Melting curve analysis of hTks5 (short) primer specificity. Shown are the superimposed analyses of the the standard curve and Hela cDNA qRT-PCR products. Low mRNA expression of Tks5 (short) was detected (~8.98 ± 0.35 copies/ng total RNA).

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In Situ Zymography Results

Figure 5. Quantitative Analysis of Gelatin Degradation Induced by the Overexpression of Each mTks5 Splice Form in LNCaP Cells. Data are mean gelatin degradation values (area units/cell) from 9-11 images analyzed for each treatment with SEM. Two-tailed Student’s t-tests revealed that Tks5-A, B, and AB induced significantly more degradation than the short form and mock treatments at the 95% level.

Figure 4. Comparison of Gelatin Degradation Induced by the Overexpression of Each mTks5 Splice Form in LNCaP Cells. Representative images of LNCaP cells transfected with the indicated constructs. NIH-3T3 cells overexpressing Src (Src-3T3) served as the positive control treatment. Degradation (dark spots) as the result of invadopodia activity by cells stained with DAPI (blue) can be seen in the Alexa Fluor 488-conjugated gelatin monolayer (green). Also shown is the immunoblot analysis of Tks5 and GAPDH expression in transfected LNCaP cells.

qRT-PCR Strategy To ensure specific Tks5 splice form mRNA detection, primers were designed to anneal inside each splice site or span the corresponding region of the shortest form. All amplicon lengths were 281-282 bp with Tm values (IDT OligoAnalyzer) ranging from 59.6-60.6°C. No significant primer dimer interactions were predicted by the ThermoFisher Multiple Primer Analyzer program using the optimal setting.

Figure 6. qRT-PCR Strategy. Combinations of two forward and two reverse primers allows for the detection of all four active Tks5 splice variants. Cloning Human Tks5 (short) was generated by deleting the second splice region from a previously constructed pcDNA3-hTks5-B plasmid. hTks5-A and AB were generated via the insertion of the first splice region into hTks5 (short) and B respectively. All insertions and deletions were performed with the NEB Q5 Site Directed Mutagenesis Kit. Primers were designed using the NEBase Changer program. Construct sequences were verified via restriction digest analysis and sequencing of the target region.

Figure 7. Cloning Strategies for Generating hTks5 Constructs. (A) hTks5 (short) was generated by deleting Splice Site B from hTks5-B. (B) hTks5-A and AB were generated by inserting Splice Site A into hTks5 (short) and B respectively.

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qRT-PCR Analysis of Tks5 Splicing

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Invadopodia are actin-rich ventral protrusions of invasive cancer cells that play an important role in tumor metastasis.1,2 These structures exert a motive force on the extracellular matrix (ECM) and release proteases required for its degradation and remodeling. Thus, invadopodia allow cancer cells to free themselves from their native environments and invade other tissues during metastasis. It is the purpose of this project to determine, in the context of prostate cancer, the effects of Tks5 splicing on invadopodia activity. Tks5 is a substrate of Src tyrosine kinase, the first discovered oncogene, and serves to scaffold the machinery required for ECM adhesion and degradation.3-5 Tks5 has two alternative splice sites (A and B) that give rise to four potential splice forms: Tks5 (short), Tks5-A, Tks5-B, and Tks5-AB. We hypothesize that the Tks5 splice forms will differentially induce invadopodia activity and thus make Tks5 alternative splicing useful for tracking the progression of prostate cancers in patients. To test this hypothesis, bacterial plasmids containing each Tks5 splice form were transfected into the LNCaP prostate cancer cell line. Immunoblotting and in situ zymography were then used to assess Tks5 expression and invadopodia activity respectively. While a previous report indicated that the short Tks5 splice form enabled invadopodia-associated matrix degradation in LNCaP cells, we now show that the longer Tks5 variants, Tks5-A (p = 0.022), B (p = 0.034), and AB (p = 0.010) induce significantly greater matrix remodeling activity. In the future, invasion assays will be used to validate the matrix remodeling data while qRT-PCR will be employed to determine if Tks5 alternative splicing is a tissue-specific phenomenon with useful diagnostic applications.

Experimental Overview Plasmid pcDNA3 vectors containing each of the four murine Tks5 splice forms were transfected into LNCaP prostate cancer cells using proprietary reagents and procedures developed by Amaxa. Transfected cells were then incubated at 37°C for 48 hours in both a 6-well plate for lysates and a 24-well plate containing Oregon Green 488-conjugated gelatin-coated coverslips for matrix degradation analysis.6 Lysates were normalized for protein and Tks5 expression was determined via SDS-PAGE and western blot using GAPDH as a reference. The coverslips were harvested and the cells were fixed, permeabilized, and mounted onto glass sides in ProLong Gold Plus with DAPI (Life Technologies). Using 9-11 images from each coverslip, the amount of gelatin degradation per cell per normalized image was determined for each treatment using Image J software.7 Src-3T3 cells were used as a positive control to assess gelatin quality.

Figure 3. In Situ Zymography Overview

Harvest cells

Incubate cells for 48 hours at 37°C in a 24-well dish containing gelatin-coated coverslips and a 6-well dish for lysates

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Normalize gelatin degradation to number of nuclei using Image J

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Abstract

In Situ Zymography

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Figure 1. Invadopodia Structure and Function in Src-3T3 Cells. (A) Invadopodia (gold arrow) form rosette superstructures (white arrow) in mature, invasive cancer cells. Actin was stained with phalloidin 594 and nuclei with DAPI. (B) Black areas correspond to invadopodia-mediated matrix degradation. Coverslips were coated with Alexa Fluor 488-conjugated gelatin to simulate extracellular matrix.

F-actin Nuclei Gelatin

Figure 2. Location of the Two Splice Sites in Tks5

PX SH3A SH3B SH3C SH3D SH3E Y

Splice Site B +/-28aa

SH3A Splice Site B Splice Site A

Splice Site A +/-15aa