- Open Access
An aberrant spliced transcript of focal adhesion kinase is exclusively expressed in human breast cancer
- Ling Yao†1,
- Kai Li†1,
- Wenting Peng1,
- Qiang Lin2,
- Shan Li1,
- Xin Hu†1,
- Xinmin Zheng3 and
- Zhiming Shao1Email author
© Yao et al.; licensee BioMed Central Ltd. 2014
- Received: 16 February 2014
- Accepted: 17 May 2014
- Published: 21 May 2014
To clarify the roles of a new aberrantly spliced transcript of FAK that lacks exon 26 (denoted -26-exon FAK) in human breast cancers.
Transcripts of FAK expressed in 102 human breast tumor tissues and 52 corresponding normal tissues were analyzed by RT-PCR and DNA sequencing, as well as agarose gel electrophoresis. The cDNA of -26-exon FAK was cloned and expressed in MCF-10A cells, and then the kinase activity, cellular localization and migration capability of FAK were examined by western blotting, immunofluorescent staining and migration assays, respectively. The expression levels of FAK were analyzed by western blotting in MCF-7 cells treated with TNF-α or in MCF-10A cells upon serum deprivation. The MCF-10A cells transfected with a plasmid expressing -26-exon FAK were cultured in serum-free medium and cell apoptosis was analyzed by flow cytometry.
The -26-exon FAK transcript was exclusively present in human breast tumor tissues and the encoded protein possessed the same kinase activity, cellular localization and cell migration-promoting ability as wild-type FAK. In MCF-7 cells treated with TNF-α, and in MCF-10A cells upon serum deprivation, the -26-exon FAK was resistant to proteolysis while wild-type FAK was largely cleaved. In addition, the -26-exon FAK, but not wild-type FAK, inhibited cell apoptosis.
The -26-exon FAK transcript, which is exclusively expressed in human breast tumor tissues, encodes a protein that possesses the same kinase activity and biological function as the wild-type FAK, but because it is resistant to the caspase-mediated cleavage that induces the proteolysis of the wild-type form, it ultimately prevents apoptosis.
- Exon 26
- Breast cancer
FAK is a non-receptor tyrosine kinase that plays a key role at focal adhesion sites by promoting cell spreading, migration, and the transmission of anchorage-dependent anti-apoptotic signals . FAK is activated via auto-phosphorylation and phosphorylation by other tyrosine kinases, including the Src family of kinases . Its auto-phosphorylation at the Tyr-397 site is an important event for maintaining the biological function of FAK, because it creates a high-affinity binding site for proteins with SH2 domains, including the Src family kinases, which will further phosphorylate FAK on other tyrosine residues, such as Tyr-576 and 577, to positively up-regulate FAK activity. The C-terminal region of FAK contains two proline-rich sequences  and also harbors a focal adhesion-targeting (FAT) sequence that associates with other proteins, including paxillin . FAK is an important mediator of cell proliferation, migration, and survival, and any perturbation of these processes is often associated with the development of malignancy. In fact, increased FAK levels have been reported in many types of cancers, including prostate, cervix, colon, ovary, and breast cancer . FAK may promote tumorigenesis by directly maintaining tumor growth , preventing apoptosis and promoting the survival of tumor cells [5–7], and modulating focal adhesion dynamics and the cellular cytoskeleton to facilitate cancer cell invasion and metastasis [8, 9].
Apoptosis plays a key role in regulating tissue development and preventing cancer metastasis . During the progression of apoptosis, the executioner caspases caspase-3 and caspase-7 are major effector caspases that can proteolyze a large number of substrates, including FAK, to accelerate apoptosis [10, 11]. Apoptosis can be initiated when adherent cells detach from the basement matrix, a process which is generally defined as “anoikis” . However, the evasion of anoikis is frequently observed in FAK-overexpressing or -mutated tumors [11, 12]. Thus, the expression level and activity of FAK is closely associated with tumorigenesis, which indicates that FAK may be an important and useful cancer marker for future cancer diagnosis and therapy [1, 2, 8, 9]. Previous studies have revealed that the post-transcriptional regulation of FAK is conserved in rodents and humans, and the pathological disturbance of alternatively spliced FAK may lead to abnormal cellular regulation and even tumorigenesis [13, 14]. Thus, we analyzed the FAK expression at the RNA level in human breast cancer, aiming to explore whether there are alternatively spliced transcripts of FAK in tumors and to dissect the roles of these FAK transcripts in tumorigenesis.
In this study, we found that an aberrantly spliced transcript of FAK kinase missing the exon 26 segment is exclusively expressed in human breast cancer and that this FAK mutant is resistant to caspase-mediated proteolysis, even though it possesses the same kinase activity as wild-type FAK. Moreover, this FAK mutant inhibited apoptosis when cultured in serum-free medium. Thus, we propose that the exon 26-deletion mutant of FAK may promote the progression of breast cancer by resisting apoptosis and promoting tumor cell survival.
RNA extraction and real-time PCR
Both breast tumor and corresponding normal tissues were acquired from the Mammary Gland Branch of the Shanghai Cancer Center. Fresh tissue samples weighing ~25 mg were homogenized twice on ice for 20 s with a PowerGen Model 125 Homogenizer (Fisher Scientific, MA, USA) in 1.5-ml microcentrifuge tubes containing 0.5 ml of the TRIzol RNA isolation reagent (Invitrogen, NY, USA) according to the manufacturer’s instructions. The cDNAs were reversely transcribed using Superscript II (Invitrogen, NY, USA), and PCR was performed using the Platinum® Taq DNA polymerase (Invitrogen, NY, USA) according to the manufacturer’s instructions. The PCR primers used are the following: FAK sense, 5’-gccttaacaatgcgtcagtttgacc; FAK antisense, 3’-tcagtgtggtctcgtctgcccaag. The PCR products were cloned using an Original TA Cloning Kit (TaKaRa, Japan), according to the manufacturer’s instructions, and five clones were then randomly selected for sequencing. The real-time PCR was performed with SYBR Premix Ex Taq™ (TaKaRa, Japan) according to the manufacturer’s instructions. The primers for segment 1 (1304 to 1416 bp) of FAK: forward, 5’-ccatccctaaccattgcg-3’; reverse, 5’-gcccgttcaccttctttct-3’. The primers for segment 2 (2546 to 2646 bp) of FAK: forward, 5’-ggctaccctggttcacat-3’; reverse, 5’-ctgccacattgctatctcct-3’. The primers for GAPDH: forward, 5’-tgggctacactgagcaccag-3’; reverse, 5’-gggtgtcgctgttgaagtca-3’.
Both the MCF-10A and MCF-7 cell lines were purchased from the American Type Culture Collection (ATCC, VA, USA). The MCF-7 cells were grown in DMEM (Gibco, NY, USA) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone, UT) supplemented with 100 μg/ml sodium pyruvate, 10 μg/ml insulin (Sigma, MO, USA), 100 units/ml penicillin, and 100 μg/ml streptomycin. The MCF-10A cells were cultured in DMEM/F12 (Invitrogen, NY, USA) containing 10% (v/v) horse serum (Invitrogen, NY, USA) supplemented with 20 ng/ml EGF (PeproTech), 0.5 mg/ml hydrocortisone (Sigma, MO, USA), 100 ng/ml cholera toxin (Sigma, MO, USA), 10 μg/ml insulin, 100 units/ml penicillin, and 100 μg/ml streptomycin. For TNF-α induction, TNF-α was used at a concentration of 50 ng/ml. For serum-free induction, the MCF-10A cells were cultured in DMEM/F12 medium containing all of the other components except serum.
Construction of gateway plasmids and virus infection
All of the following Gateway reagents and vectors were purchased from Invitrogen, and all of the procedures were performed according to the manufacturer’s instructions. Briefly, the full-length cDNA encoding wild-type or mutated FAK with a 5’ sequence encoding three HA epitopes (YPYDVPDYA) was generated by PCR and integrated into the pDONR™201 vector through a BP reaction to form the entry clones. The correct entry clones were then used for LR recombination with the pLenti6/V5-DEST lenti-virus-expressing vector. The successfully cloned pLenti-DEST constructs were transfected into 293 T cells using the ViraPower™ Lenti-viral Packaging Mix to produce the lenti-virus. FAK expression was detected by Western blot, and the lenti-virus was amplified and saved for further use.
Immunoprecipitation and Western blot
The cells were lysed in lysis buffer (50 mM Tris-Cl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, protease and phosphatase inhibitor cocktail). For immunoprecipitation, 500 μg of the total cell extract from each sample was used. The extracts were incubated with 10 μl of HA antibody (Sigma, MO, USA) overnight at 4°C. Then, 30 μl of protein A/G-agarose beads (Santa Cruz, CA, USA) were added, and the mixture was incubated with rocking for 2 h at 4°C. The precipitates were washed three times with lysis buffer, resuspended in 40 μl of 1× sample buffer, and then boiled at 100°C for 10 min. The Western blot was performed according to the protocols supplied with each antibody. The following antibodies were used in this study: anti-FAK (BD Biosciences, 1:2000 dilution), anti-Tyr-397 (Cell Signaling, 1:1000 dilution), anti-Tyr-576/577 (Cell Signaling, 1:1000 dilution), anti-caspase-3 (Cell Signaling, 1:1000 dilution), and anti-caspase-7 (Cell Signaling, 1:1000 dilution). The intensity of the bands was analyzed using the ImageJ software (NIH Image).
Immunofluorescent staining and confocal microscopy
The cells were plated onto coverslips coated with 50 μg/ml bovine plasma fibronectin (Sigma, MO, USA) and cultured at 37°C for 16 h. The cells were then fixed in 3.7% paraformaldehyde in 0.01 M PBS for 10 min and permeabilized with 0.01% Triton X-100 for 10 min on ice. After extensive washing with 0.01 M PBS, the cells were blocked with 5% BSA in 0.01 M PBS for 30 min at room temperature and then incubated with primary HA antibody (1:100 dilution) overnight at 4°C. After washing three times with 0.01 M PBS, the cells were incubated with the rhodamine-labeled secondary antibody (1:400 dilution) for 1 h and DAPI (Roche, Nutley, NJ, USA) for 1 min at room temperature and then visualized under a confocal microscope according to the manufacturer’s instructions.
MCF-10A cells transfected with wild-type or -26-exon FAK constructs were plated onto the upper membrane of transwells (8-μm pore size, Millipore, MA) at a density of 4 × 105 cells for per well and cultured for 12 h. Any non-migrated cells on the upper membrane were removed with a cotton swab, and the migrated cells (located on the lower surface of the filters) were fixed for 5 min in methanol, stained with 0.1% crystal violet, eluted with 33% ethylic acid, and measured at 570 nm to obtain their OD values. The experiment was repeated three times.
Flow cytometry analysis
For the flow cytometry analysis, MCF-10A cells at a density of 2 × 105/ml were doubly labeled with PI and Annexin V-FITC in the dark at room temperature according to the instructions provided by the Annexin V-FITC/PI kit (Invitrogen, NY, USA). Each analysis was repeated three times.
All of the human tissues were acquired from the Mammary Gland Branch of the Shanghai Cancer Center, Shanghai Medical College, Fudan University. Written informed consent was provided by all of the patients, and the protocols were performed in accordance with approval from the Ethic Committee of Fudan University Shanghai Cancer Center (Permit Numbers 050432-4-1008A).
All of the data were analyzed using the SPSS 13.0.0 (SPSS Inc., Chicago, IL, USA) software, and the comparisons between two groups were performed using Student’s t-test. Values of P < 0.05 were considered significant and are indicated by asterisks in the figures.
The -26-exon FAK protein is exclusively expressed in tumor tissues
The -26-exon FAK protein has the same characteristics as the wild-type FAK
The -26-exon FAK protein is resistant to caspase-mediated proteolysis
The -26-exon FAK protein promotes cell survival
The region composed of amino acids 744 to 789 is absent in the -26-exon-deletion FAK and has not been reported to interact with other proteins. Thus, we first attempted to explore the kinase activity and other profiles of -26-exon FAK, and the results showed that this protein acts similarly to wild-type FAK. Previous studies showed that FAK is a substrate of caspases [11, 12], and we excitingly found that a caspase-sensitive cleavage site is located in this absent fragment, which prompted us to examine the degradation status of -26-exon FAK during apoptosis. This study revealed that -26-exon FAK is indeed resistant to proteolysis by caspase. Although several other caspase-mediated cleavage sites were also found in FAK [11, 12], in our study the wild-type FAK was largely proteolyzed, whereas the -26-exon FAK protein resisted proteolytic degradation, indicating that the absent region may be the main, or the most sensitive, region of FAK that can be cleaved by caspases during apoptosis. It is worth noting that the exon 26 deletion is often associated with the mutation of Leu to Pro at position 961, which is localized on the C-terminus of FAK. However, the actual function of this mutation remains to be elucidated.
FAK is essential for focal adhesion and cell migration and harbors anti-apoptosis capability. Adherent cells that are deprived of the extracellular matrix initiate the apoptotic process, during which FAK is cleaved and degraded by activated caspases. However, the -26-exon FAK mutant is resistant to cleavage and exerts its anti-apoptosis effect to promote tumor cell survival (Figure 4C). Thus, we hypothesize that -26-exon FAK may help tumor cells evade apoptosis and promote tumor cell survival during the process of cancer metastasis when the outer plate of tumor cells is deprived of cancer-foci and ready for metastasis. In addition, the notably down-regulated Akt activation in the wild-type group compared with the -26-exon FAK group after treatment with serum-free medium for 12 h (Figure 3C and D) at least partially explains the phenomenon that -26-exon FAK and not wild-type FAK can promote cell survival in serum-free medium, which is in agreement with the previous finding that FAK may promote cell survival via the PI3K/Akt pathway . In addition, FAK is a key regulator of cell migration; thus, the caspase-resistant -26-exon FAK may efficiently promote the invasion of cancer cells, thereby accelerating the process of tumor metastasis.
To explore whether -26-exon FAK expresses in available breast tumor cell lines such as BT-474, MCF-7, the percentage of -26-exon FAK expression was determined using the strategy as described in Figure 1D and E. It is interesting to find that the signals of -26-exon FAK expression were observed in breast tumor cell line BT-549 and HCC1937 (Additional file 1: Figure S4). The results indicate that -26-exon FAK, the aberrant transcript of FAK may be widely expressed in a variety of breast tumors, and may become a new marker of breast cancer. In addition, the breast tumor cell line BT-549 and HCC1937 may be the useful cell lines for future studies in clarifying the roles of -26-exon FAK in tumorigenesis. The histological grading of 6 samples expressing the -26-exon FAK was II ~ III, and the TNM staging was II ~ III (Additional file 2: Table S1). And 5 out of 6 samples had lymph node metastasis. Recurrence of the breast tumor was observed in the Sample-1 patient 2 years after the surgery; pulmonary metastasis was observed in the Sample-5 patient 2 years after the surgery; the Sample-6 patient died from brain metastasis 1 year after the surgery. From these results, we speculated that the expression of -26-exon FAK may correlate with the tumor degree or metastasis to some extent.
Taken together, the above results indicate that the alternatively spliced transcript -26-exon FAK is exclusively expressed in tumor tissues, efficiently promotes tumor cell survival by preventing apoptosis, and may be extremely useful as a new marker for future cancer diagnosis.
We acknowledge American Journal Experts for significant revision of the manuscript. This work was supported by the National Natural Science Foundation of China (No 81202082).
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