Limits...
Fas Activated Serine-Threonine Kinase Domains 2 (FASTKD2) mediates apoptosis of breast and prostate cancer cells through its novel FAST2 domain.

Das S, Yeung KT, Mahajan MA, Samuels HH - BMC Cancer (2014)

Bottom Line: Although FASTKD2 is an inner mitochondrial membrane protein, it does not require mitochondrial localization to initiate apoptosis.We found that the pathway leads to apoptosis in LNCaP cells, including the two androgen-independent LNCaP cell lines that are generally resistant to apoptosis.Lastly, we identified that FASTKD2-mediated apoptosis is initiated by the 81 amino acid FAST2 region.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, PHL 814, New York University School of Medicine, 455 First Ave,, New York, NY 10016, USA. herbert.samuels@nyumc.org.

ABSTRACT

Background: Expression of NRIF3 (Nuclear Receptor Interacting Factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. This occurs through binding of NRIF3 or its 30 amino acid Death Domain-1 (DD1) region to the transcriptional repressor, DIF-1 (DD1 Interacting Factor-1). DIF-1 acts in a wide variety of breast cancer cells but not other cell types to repress the pro-apoptotic gene, FASTKD2. Expression of NRIF3 or DD1 inactivates the DIF-1 repressor leading to rapid derepression of FASTKD2, which initiates apoptosis within 5-8 h of expression. Although FASTKD2 is an inner mitochondrial membrane protein, it does not require mitochondrial localization to initiate apoptosis.

Methods: Androgen dependent LNCaP cells as well as two androgen independent LNCaP cell lines (LNCaP-AI and LNCaP-abl) were studied and LNCaP-AI cells were engineered to conditionally express DD1 or the inactive DD1-S28A with 4-hydroxytamoxifen. Apoptosis was assessed by TUNEL assay. FASTKD2 is related to 4 other proteins encoded in the human genome (FASTKD1, 3, 4, 5). All contain a poorly conserved putative bipartite kinase domain designated as FAST1_FAST2. We examined whether expression of any of the other FASTKD isoforms leads to apoptosis and sought to identify the region of FASTKD2 necessary to initiate the apoptotic pathway.

Results: Of the FASTKD1-5 isoforms only expression of FASTKD2 leads to apoptosis. Although, the NRIF3/DD1/DIF-1 pathway does not mediate apoptosis of a wide variety of non-breast cancer cell lines, because of certain similarities and gene signatures between breast and prostate cancer we explored whether the NRIF3/DD1/DIF-1/FASTKD2 pathway mediates apoptosis of prostate cancer cells. We found that the pathway leads to apoptosis in LNCaP cells, including the two androgen-independent LNCaP cell lines that are generally resistant to apoptosis. Lastly, we identified that FASTKD2-mediated apoptosis is initiated by the 81 amino acid FAST2 region.

Conclusions: The NRIF3/DIF-1/FASTKD2 pathway acts as a "death switch" in breast and prostate cancer cells. Deciphering how this pathway is regulated and how FASTKD2 initiates the apoptotic response will allow for the development of therapeutic agents for the treatment of androgen-independent prostate cancer or Tamoxifen-unresponsive Estrogen Receptor negative tumors as well as metastatic breast or prostate cancer.

Show MeSH

Related in: MedlinePlus

Activation of DD1 leads to expression of FASTKD2 which mediates the apoptotic response. (A) LNCaP-AI cells stably expressing DD1-ERT2 or DD1(S28A)-ERT2 were incubated with 1 uM 4-OHT for 8 h and then analyzed for DD1 and DD1(S28A) expression by immunofluorescense using FLAG-M2 antibody (green) and apoptosis by TUNEL assay (red). Nuclei were stained with DAPI (blue). (B) LNCaP-AI cells stably expressing DD1-ERT2 were transfected with a Control siRNA or an siRNA known to knock down expression of FASTKD2 [5]. Twenty-four h later the cells were transfected again with the siRNAs as described in the Methods section. Twenty h after the second siRNA transfection cells were incubated with 4-OHT for 15 h. Cells were then fixed and permeablized for TUNEL assay (red). Nuclei are stained with DAPI (blue). Cells treated with the FASTKD2 siRNA exhibited essentially no apoptosis after 4-OHT incubation while cells treated with the Control siRNA showed extensive apoptosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4256816&req=5

Fig3: Activation of DD1 leads to expression of FASTKD2 which mediates the apoptotic response. (A) LNCaP-AI cells stably expressing DD1-ERT2 or DD1(S28A)-ERT2 were incubated with 1 uM 4-OHT for 8 h and then analyzed for DD1 and DD1(S28A) expression by immunofluorescense using FLAG-M2 antibody (green) and apoptosis by TUNEL assay (red). Nuclei were stained with DAPI (blue). (B) LNCaP-AI cells stably expressing DD1-ERT2 were transfected with a Control siRNA or an siRNA known to knock down expression of FASTKD2 [5]. Twenty-four h later the cells were transfected again with the siRNAs as described in the Methods section. Twenty h after the second siRNA transfection cells were incubated with 4-OHT for 15 h. Cells were then fixed and permeablized for TUNEL assay (red). Nuclei are stained with DAPI (blue). Cells treated with the FASTKD2 siRNA exhibited essentially no apoptosis after 4-OHT incubation while cells treated with the Control siRNA showed extensive apoptosis.

Mentions: To further study the mechanism of DD1-mediated apoptosis of LNCaP cells, we generated stable LNCaP-AI cell lines expressing DD1-ERT2 or DD1(S28A)-ERT2. ERT2 is a mutated form of the human estrogen receptor-α ligand binding domain that does not bind estrogen agonists but binds the partial agonist–antagonist 4-hydroxytamoxifen (4-OHT) [19]. Both DD1-ERT2 and DD1(S28A)-ERT2 are expressed with an N-terminal nuclear localization signal and a FLAG epitope to allow for estimation of expression and cell distribution. Without 4-OHT, DD1-ERT2 and DD1(S28A)-ERT2 are sequestered in a heat shock protein complex mostly outside the nucleus. After 4-OHT incubation the chimeric proteins rapidly enter the nucleus. In addition, 4-OHT stabilizes the chimeric protein further increasing its level of expression in the cell. Such studies in breast cancer cells indicate that after 4-OHT incubation with DD1-ERT2 expressing cells, apoptosis (TUNEL) is detected within 4 h and is maximal between 5–10 h [5]. No apoptosis was found with the breast cancer lines expressing DD1(S28A)-ERT2 after 4-OHT incubation [5]. Figure 3A shows such a study in LNCaP-AI cells stably expressing DD1-ERT2 or DD1(S28A)-ERT2. Eight h after 4-OHT incubation the DD1-ERT2 LNCaP-AI cells exhibit extensive apoptosis by TUNEL assay while the DD1(S28A)-ERT2 LNCaP-AI cells are TUNEL negative.Figure 3


Fas Activated Serine-Threonine Kinase Domains 2 (FASTKD2) mediates apoptosis of breast and prostate cancer cells through its novel FAST2 domain.

Das S, Yeung KT, Mahajan MA, Samuels HH - BMC Cancer (2014)

Activation of DD1 leads to expression of FASTKD2 which mediates the apoptotic response. (A) LNCaP-AI cells stably expressing DD1-ERT2 or DD1(S28A)-ERT2 were incubated with 1 uM 4-OHT for 8 h and then analyzed for DD1 and DD1(S28A) expression by immunofluorescense using FLAG-M2 antibody (green) and apoptosis by TUNEL assay (red). Nuclei were stained with DAPI (blue). (B) LNCaP-AI cells stably expressing DD1-ERT2 were transfected with a Control siRNA or an siRNA known to knock down expression of FASTKD2 [5]. Twenty-four h later the cells were transfected again with the siRNAs as described in the Methods section. Twenty h after the second siRNA transfection cells were incubated with 4-OHT for 15 h. Cells were then fixed and permeablized for TUNEL assay (red). Nuclei are stained with DAPI (blue). Cells treated with the FASTKD2 siRNA exhibited essentially no apoptosis after 4-OHT incubation while cells treated with the Control siRNA showed extensive apoptosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4256816&req=5

Fig3: Activation of DD1 leads to expression of FASTKD2 which mediates the apoptotic response. (A) LNCaP-AI cells stably expressing DD1-ERT2 or DD1(S28A)-ERT2 were incubated with 1 uM 4-OHT for 8 h and then analyzed for DD1 and DD1(S28A) expression by immunofluorescense using FLAG-M2 antibody (green) and apoptosis by TUNEL assay (red). Nuclei were stained with DAPI (blue). (B) LNCaP-AI cells stably expressing DD1-ERT2 were transfected with a Control siRNA or an siRNA known to knock down expression of FASTKD2 [5]. Twenty-four h later the cells were transfected again with the siRNAs as described in the Methods section. Twenty h after the second siRNA transfection cells were incubated with 4-OHT for 15 h. Cells were then fixed and permeablized for TUNEL assay (red). Nuclei are stained with DAPI (blue). Cells treated with the FASTKD2 siRNA exhibited essentially no apoptosis after 4-OHT incubation while cells treated with the Control siRNA showed extensive apoptosis.
Mentions: To further study the mechanism of DD1-mediated apoptosis of LNCaP cells, we generated stable LNCaP-AI cell lines expressing DD1-ERT2 or DD1(S28A)-ERT2. ERT2 is a mutated form of the human estrogen receptor-α ligand binding domain that does not bind estrogen agonists but binds the partial agonist–antagonist 4-hydroxytamoxifen (4-OHT) [19]. Both DD1-ERT2 and DD1(S28A)-ERT2 are expressed with an N-terminal nuclear localization signal and a FLAG epitope to allow for estimation of expression and cell distribution. Without 4-OHT, DD1-ERT2 and DD1(S28A)-ERT2 are sequestered in a heat shock protein complex mostly outside the nucleus. After 4-OHT incubation the chimeric proteins rapidly enter the nucleus. In addition, 4-OHT stabilizes the chimeric protein further increasing its level of expression in the cell. Such studies in breast cancer cells indicate that after 4-OHT incubation with DD1-ERT2 expressing cells, apoptosis (TUNEL) is detected within 4 h and is maximal between 5–10 h [5]. No apoptosis was found with the breast cancer lines expressing DD1(S28A)-ERT2 after 4-OHT incubation [5]. Figure 3A shows such a study in LNCaP-AI cells stably expressing DD1-ERT2 or DD1(S28A)-ERT2. Eight h after 4-OHT incubation the DD1-ERT2 LNCaP-AI cells exhibit extensive apoptosis by TUNEL assay while the DD1(S28A)-ERT2 LNCaP-AI cells are TUNEL negative.Figure 3

Bottom Line: Although FASTKD2 is an inner mitochondrial membrane protein, it does not require mitochondrial localization to initiate apoptosis.We found that the pathway leads to apoptosis in LNCaP cells, including the two androgen-independent LNCaP cell lines that are generally resistant to apoptosis.Lastly, we identified that FASTKD2-mediated apoptosis is initiated by the 81 amino acid FAST2 region.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, PHL 814, New York University School of Medicine, 455 First Ave,, New York, NY 10016, USA. herbert.samuels@nyumc.org.

ABSTRACT

Background: Expression of NRIF3 (Nuclear Receptor Interacting Factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. This occurs through binding of NRIF3 or its 30 amino acid Death Domain-1 (DD1) region to the transcriptional repressor, DIF-1 (DD1 Interacting Factor-1). DIF-1 acts in a wide variety of breast cancer cells but not other cell types to repress the pro-apoptotic gene, FASTKD2. Expression of NRIF3 or DD1 inactivates the DIF-1 repressor leading to rapid derepression of FASTKD2, which initiates apoptosis within 5-8 h of expression. Although FASTKD2 is an inner mitochondrial membrane protein, it does not require mitochondrial localization to initiate apoptosis.

Methods: Androgen dependent LNCaP cells as well as two androgen independent LNCaP cell lines (LNCaP-AI and LNCaP-abl) were studied and LNCaP-AI cells were engineered to conditionally express DD1 or the inactive DD1-S28A with 4-hydroxytamoxifen. Apoptosis was assessed by TUNEL assay. FASTKD2 is related to 4 other proteins encoded in the human genome (FASTKD1, 3, 4, 5). All contain a poorly conserved putative bipartite kinase domain designated as FAST1_FAST2. We examined whether expression of any of the other FASTKD isoforms leads to apoptosis and sought to identify the region of FASTKD2 necessary to initiate the apoptotic pathway.

Results: Of the FASTKD1-5 isoforms only expression of FASTKD2 leads to apoptosis. Although, the NRIF3/DD1/DIF-1 pathway does not mediate apoptosis of a wide variety of non-breast cancer cell lines, because of certain similarities and gene signatures between breast and prostate cancer we explored whether the NRIF3/DD1/DIF-1/FASTKD2 pathway mediates apoptosis of prostate cancer cells. We found that the pathway leads to apoptosis in LNCaP cells, including the two androgen-independent LNCaP cell lines that are generally resistant to apoptosis. Lastly, we identified that FASTKD2-mediated apoptosis is initiated by the 81 amino acid FAST2 region.

Conclusions: The NRIF3/DIF-1/FASTKD2 pathway acts as a "death switch" in breast and prostate cancer cells. Deciphering how this pathway is regulated and how FASTKD2 initiates the apoptotic response will allow for the development of therapeutic agents for the treatment of androgen-independent prostate cancer or Tamoxifen-unresponsive Estrogen Receptor negative tumors as well as metastatic breast or prostate cancer.

Show MeSH
Related in: MedlinePlus