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. 2019 Aug;21(8):837-848.
doi: 10.1016/j.neo.2019.05.008. Epub 2019 Jul 12.

The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of Its Cell Surface Expression and Viability of Tamoxifen-Resistant Breast Cancer Cells

Chun-Chih Tseng  1 , Pu Zhang  2 , Amy S Lee  3
Affiliations

The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of Its Cell Surface Expression and Viability of Tamoxifen-Resistant Breast Cancer Cells

Chun-Chih Tseng et al. Neoplasia. 2019 Aug.

Abstract

Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

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Figures

Figure 1
Figure 1
GRP78 colocalizes with CD44v in MCF7-LR breast cancer cells. (A) Immunofluorescence and confocal images showing the distribution and colocalization of GRP78 (red) and CD44v (green) in the saponin-permeabilized MCF7-LR breast cancer cells. GRP78 and CD44v were detected by MAb159 and anti-CD44v3 antibodies, respectively. The mouse-on-mouse (M.O.M.) control staining was performed with the same protocol as double-stained cells but lacking the primary antibody targeting CD44v. No primary control staining was conducted without the primary antibodies but with secondary antibodies. The nuclei were stained by DAPI in blue. Thickness of single immunofluorescent image section: 0.3 μm. Signals were visualized with 63×/1.4 NA objective on a Leica TCS SP8 confocal microscope. Scale bars, 20 μm. (B) Immunofluorescence and confocal images showing the distribution and colocalization of GRP78 (red) and CD44v (green) on the cell surface of nonpermeabilized MCF7-LR breast cancer cells. GRP78 and CD44v were detected by MAb159 and anti-CD44v3 antibodies, respectively. The M.O.M. control staining was performed with the same protocol as double-stained cells but lacking primary antibody targeting CD44v. No primary control staining was conducted without the primary antibodies but with secondary antibodies. The nuclei were stained by DAPI in blue. DIC: differential interference contrast. Thickness of single immunofluorescent image section: 0.8 μm. Cell peripheries were outlined with black lines. Signals were visualized with 100×/1.4 NA objective on an LSM 510 confocal microscope. Scale bars, 20 μm.
Figure 2
Figure 2
The COOH-terminal proline-rich region of GRP78 is essential for forming complex with CD44v in vitro. (A) Schematic representation of the human GST-tagged GRP78 wild-type and deletion mutants cloned into pGEX-4T-1 backbone vector. a.a., amino acids. FL, a.a. 19-654; N, a.a. 19-407; C, a.a. 413-654; ΔKDEL, a.a. 19-650; ΔC11, a.a. 19-643; ΔC17, a.a. 19-637; ΔC73, a.a. 19-581; C73, a.a. 582-654. The locations of the ER signal, ATPase domain, substrate binding domain, proline-rich region, and KDEL motif of GRP78 are depicted on top. (B) Schematic representation of the expression construct of HA-tagged human CD44 containing variable exon 3 to 10. EC, extracellular; TM, transmembrane; IC, intracellular. (C-D) Western blot analysis of samples from in vitro GST pull-down assay. GST or GST-tagged GRP78 wild-type and mutant proteins purified from E. coli (BL21) were incubated with 293T whole cell lysate containing overexpressed CD44v-HA (vHA). (E) Upper panel: I-TASSER model of full-length human GRP78 protein. ATPase domain is in light blue. SBD is in orange. The last 27 amino acids close to the COOH-terminus (C27) are lighted in rainbow color and by dashed circle. Arrow indicates the PPP polyproline amino acids (red). Lower panel: The enlarged C27 area shows the polyproline amino acids (a.a. 640-642; red) and the adjacent protein structure.
Figure 3
Figure 3
The COOH-terminal polyproline sequence of GRP78 is critical for its cell surface expression and modulates STAT3 signaling in MCF7-LR breast cancer cells. (A) Schematic representation of FLAG-tagged human GRP78 (F-GRP78) wild-type (WT) and mutant harboring a PPP to AAA mutation (AAA). a.a., amino acids. SBD, substrate binding domain. (B) Western blot analysis of MCF7-LR cells transfected with FLAG-tagged GRP78 WT, AAA, or pcDNA3 backbone vector (v). Annexin II and β-actin are loading controls for cell surface fraction and whole cell lysate, respectively. (C) Quantitative analysis of cell surface (cs) F-GRP78 levels. Data represent means ± SEM from three biological repeats. **P < .01 by Student's t test. (D) Schematic illustration of siRNA designed to target 3′ UTR of Grp78. WT and AAA expression plasmids do not contain 3′ UTR. (E) Western blot analysis of MCF7-LR cells co-transfected with v, WT, or AAA and control (sictrl) or Grp78 siRNA targeting 3′ UTR (siGrp78, 3′ UTR). Numbers below the β-actin bands represent individual experimental conditions. (F) Quantification of the ratio of pSTAT3 (Y705) and STAT3 of each experimental condition as shown in panel E. Data represent means ± SEM from three biological repeats. *P ≤ .05 by Student's t test.
Figure 4
Figure 4
Comparisons of the proline-rich regions of GRP78. (A) I-TASSER model of human GRP78 (a.a. 413-654) demonstrating the three-dimentional localizations of PPP and IPPAP/PQ proline-rich sequences. The classical IPPAP motif was highlighted in magenta, the PQ amino acids in blue, and the COOH-terminal PPP polyproline amino acids in red. (B) Results of multiple sequence alignment of GRP78 COOH-terminal proline-rich regions from a broad range of species using MEGA-X software with polyproline residues in red. DnaK is a bacterial homolog of GRP78. Asterisk indicates conserved residue among all species. Dots show conserved residues with human (Homo sapiens) GRP78. Dashes indicate gaps. (C) Results of multiple sequence alignment of GRP78 IPPAP/PQ proline-rich regions from a broad range of species using MEGA-X software with the proline residues localized at the classical IPPAP motif in magenta and the proline residue localized at the PQ amino acids in blue. DnaK is a bacterial homolog of GRP78. Asterisks indicate conserved residues among all species. Dots show conserved residues with human GRP78. (D) The COOH-terminal amino acid sequences of other human (H. sapiens) heat shock proteins with polyproline residues in red.
Figure 5
Figure 5
Enforced expression of short peptide encoding the proline-rich region of GRP78 reduced cell viability and promoted apoptosis in MCF7-LR breast cancer cells. (A) Schematic illustration of expression plasmids containing wild-type or mutant sequences encoding 20 amino acids spanning the proline-rich region of GRP78 (a.a. 640-642). The sequences were preceded by a secretory and sorting peptide sequence of α-melanocyte-stimulating hormone (SSP). The plasmids were constructed in pcDNA3 backbone vector (v). P, the plasmid containing wild-type proline-rich sequence. mP, the plasmid containing PPP to AAA mutation. (B) Schematic representation of experimental procedures. cM, conditional media. h, hours. (C) Bright-field images of MCF7-LR cells with the enforced expression of v, P, or mP. Images were photographed 48 hours post transfection. Scale bar, 50 μm. (D) Left: Western blot analysis of whole cell lysates from MCF7-LR cells transfected with v, P, or mP. Cells lysates were collected 48 hours posttransfection. CD44v and GRP78 were detected by anti-CD44v3 and MAb159 antibodies, respectively. β-Actin is loading control. Right: Quantification of CD44v levels compared to the control. Data represent means ± SEM from three biological repeats. *P ≤ .05 by Student's t test. (E) Quantitative analysis of cell viability by WST-1 assay in MCF7-LR cells overexpressing v, P, or mP. Cells were analyzed 48 hours posttransfection. Data represent means ± SEM from four experimental repeats. **P < .01 ***P < .001 by Student's t test. (F) Left: Western blot analysis of whole cell lysates from MCF7-LR cells treated with cM collected from MCF7-LR cells transfected with v, P, or mP. Conditional media were collected 48 hours posttransfection and then applied to new cells for another 48 hours. CD44v and GRP78 were detected by anti-CD44v3 and MAb159 antibodies, respectively. β-Actin is loading control. Right: Quantification of CD44v levels compared to the control. Data represent means ± SEM from three biological repeats. *P ≤ .05 by Student's t test.
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