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. 2024 Jul 15;20(10):3923-3941.
doi: 10.7150/ijbs.87415. eCollection 2024.

tRF-27 competitively Binds to G3BPs and Activates MTORC1 to Enhance HER2 Positive Breast Cancer Trastuzumab Tolerance

Yaozhou He  1 , Yincheng Liu  1 , Jue Gong  1 , Fan Yang  1 , Chunxiao Sun  1 , Xueqi Yan  1 , Ningjun Duan  1 , Yijia Hua  1 , Tianyu Zeng  1 , Ziyi Fu  1 , Yan Liang  1 , Wei Li  1 , Xiang Huang  1 , Jinhai Tang  2 , Yongmei Yin  1
Affiliations

tRF-27 competitively Binds to G3BPs and Activates MTORC1 to Enhance HER2 Positive Breast Cancer Trastuzumab Tolerance

Yaozhou He et al. Int J Biol Sci. .

Abstract

About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27.tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab.

Keywords: G3BPs; HER2-positive breast cancer; MTORC1; Trastuzumab resistance; tRNA-derived fragment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
tRF-27-ZDXPHO53KSN predicted trastuzumab resistance of breast cancer. A. RNA sequencing was used to measure the expression levels of tRNA-derived fragments in cell lines HBL-100, SKBR3, and JIMT1. The top 20 significant tRNA-derived fragments were selected to draw a heatmap. B. QRT-PCR analysis of tRF-30-JZOYJE22RR33, tRF-27-ZDXPHO53KSN, tRF-26-XIP2801MK8E, tRF-29-IYEVFMD0SR1Z, tRF-22-8B8871K92, and tRF-30-SERXPIN2NYDR expression in HBL-100, SKBR3 and JIMT-1 cells. Gene expression was normalized to that of U6 expression, and fold changes by comparing gene expression levels to those in HBL-100 cells were calculated using the 2 -△△Ct method. C. Serum was sampled from 28 trastuzumab-sensitive patients and 29 trastuzumab-resistant patients, total RNA was extracted, and the expression levels of tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN were measured with qRT-PCR. D. A ROC curve analysis showed the ability of tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN to predict trastuzumab resistance. E. Progression-free survivals (PFSs) in the 52 metastatic HER-2 positive breast cancer patients who received advanced trastuzumab treatment. Patients with high expression of tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN showed worse PFSs. F. tRF-30-JZOYJE22RR33, tRF-22-8B8871K92, tRF-26-XIP2801MK8E and tRF-27-ZDXPHO53KSN originate from the same digestion site of tRNA-cysgca.
Figure 2
Figure 2
tRF-27 affected trastuzumab resistance of HER2 positive breast cancer cells. A. The expression levels of tRF-27 in trastuzumab-sensitive SKBR3 cells and trastuzumab-resistant JIMT1 cells under trastuzumab stimulation by qRT-PCR. Data were expressed as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001. B. CCK-8 was performed to detect cell growth and survival. Under the stimulation of trastuzumab, the growth rate of SKBR3 cells decreased significantly, but less significantly in JIMT1 cells. Stimulated by trastuzumab, the growth rate of cells overexpressing tRF-27 increased, and knocking down tRF-27 inhibited cell growth. C. Cell colony formation assay was performed. The colony-forming ability of cells was positively correlated with the expression level of tRF-27. D. EdU assay was used to illustrate the proliferative state of cells. Overexpression of tRF-27 in both JIMT1 and SKBR3 cell lines significantly increased the proliferation, while knocking down tRF-27 inhibited this proliferation. E. Tumor xenograft in nude mice. 2*10^7 tumor cells were injected subcutaneously into every left groin of BALB-c(nude) mice which were divided into 4 groups (n=4 in each group), including groups receiving wild-type SKBR3 cells (control), SKBR3 cells overexpressing tRF-27 (tRF-27 OE), SKBR3 cells expressing blank control (tRF-27 NC), and SKBR3 cells knocking down tRF-27 (tRF-27 IN). At day 7 of subcutaneous tumorization, the latter 3 groups were injected with trastuzumab. Tumors were collected at day 28 and one tumor disappeared in one mouse of the tRF-27 NC group, and one tumor disappeared in one mouse of the tRF-27 IN group. F - G. Regularly monitoring of tumor size (mm), and tumor weigh (mg). Trastuzumab greatly inhibited tumor growth. High expression levels of tRF-27 enhanced tumor resistance.
Figure 3
Figure 3
tRF-27 bound to G3BPs and functioned independent of stress granules. A. SKBR3, JIMT1 and BT474 cells were used for tRF-27 RNA pull-down assay. Silver-stained bands of JIMT1 cells. B. Intersection between SKBR3, JIMT1 and BT474 after respectively removing the intersections in the tRF-27 group (tRF-27) and the scramble RNA group (Scramble) in the mass spectrometry results. Only one protein G3BP2 was bound simultaneously in all three cell lines. C. We tested G3BPs expression with Western blotting. In SKBR3 and JIMT1 cells, both G3BP1 and G3BP2 could bind to tRF-27. D. RNA immunoprecipitation (RIP) assay with antibodies of G3BP1 and G3BP2. tRF-27 bound to G3BP1/G3BP2 in JIMT1 and SKBR3 cells confirmed by qRT-PCR. E. Immunofluorescence assay of G3BP2 in SKBR3 cells; stress granules formed in a nutrient-deficient environment (Positive control), while the trastuzumab group did not show nucleated stress granules (Trastuzumab treatment). F. Co-immunoprecipitation (co-IP) with G3BPs; SPAG5 did not bind to G3BPs. G. Overexpressing (tRF-27 OE) and knocking down (tRF-27 IN) tRF-27 did not alter the expression level of G3BPs. H. Knockdown of G3BP2 and the expression levels of G3BP1 in cells. I. Another RNA pull-down assay was performed with G3BP2 knockdown cells; tRF-27 still bound to G3BP1.
Figure 4
Figure 4
tRF-27 bound to G3BPs through a specific structure. A. We transfected tRF-27 with 5'-FAM into SKBR3 cells, and G3BP2 protein was immunofluorescence-stained; both were predominantly located in the cytoplasm. B. Co-IP with G3BP1/G3BP2 was performed; G3BPs combined with LAMP1, RPTOR and Tuberin. C. The sequences of the probes designed for RNA pull-down assay. D. Trastuzumab-stimulated cells were collected for pull-down assays with designed probes. Loss of the middle 9 bases damaged the ability of the probes to bind to G3BP2. E. The protein structure of G3BP2. We constructed plasmids with HA-TAG tags expressing G3BP2 that lost these three domains. F.Full-length and truncated G3BP2 do not affect the expression levels of G3BP1 and LAMP1. G. Plasmids of full-length and truncated G3BP2 were transfected into HEK293T cells. tRF-27 pull-down assay was performed. tRF-27 did not bind to G3BP2 protein that lost the NTF2 domain. H. Co-IP showed that LAMP1 was also integrated into NTF2 domain. The binding between G3BP1 and G3BP2 was more likely to depend on the RGG domain. I. The protein structure of G3BP1. We constructed plasmids with HA-TAG tags expressing G3BP1 that lost these three domains. J. Full-length and truncated G3BP1 do not affect the expression levels of G3BP2 and LAMP1. K. tRF-27 did not bind to the G3BP2 protein that is missing the NTF2 or RRM domain.
Figure 5
Figure 5
tRF-27 binds to G3BP2 protein in vitro. Purified full-length and truncated HA-G3BP2 proteins. B. RNA-EMSA was performed to demonstrate the ability of RNA to bind to proteins in vitro. tRF-27 binds to the purified G3BP2 and other truncated recombinant proteins, but not to protein that lack the NTF2 domain.
Figure 6
Figure 6
tRF-27 affected the binding of G3BPs with LAMPs. A. We synthesized tRF-27 and fragments of scramble RNA, and incubated two excess RNAs (10 nmol) with equal HEK-293 cell lysate (200 μl of cell lysate, 2*10^7 cells) for 2 h, respectively. Then, the G3BP2 antibody attached to protein A/G magnetic beads were used to perform co-IP, and the protein that could bind to G3BP2 was detected by Western blotting. B-C. Silver-stained bands in co-IP. G3BP2 binding to LAMP1 was significantly reduced in lysates co-incubated with tRF-27, compared to that in the scramble RNA group. D. We repeated the experiment three times, measured and compared the band gray values, and determined that the tRF-27 group had a significant decrease in the expression of LAMP1, compared to the scramble RNA group. Data were shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001. E. Transmembrane structure of LAMP1. F-H.We used HDOCK to predict the binding of LAMP1 and tRF-27 to the NTF2 domain in G3BPs, and the results were demonstrated with PyMol. I-J. The expression of tRF-27 did not affect the expression levels of G3BPs and LAMP1. However, when the expression of tRF-27 increased, the binding of G3BP2 with LAMP1 significantly decreased. The experiment was repeated three times, the band gray values were measured and compared.
Figure 7
Figure 7
tRF-27 promoted the activation of MTORC1. A-B. The expression levels of PI3K/AKT/mTOR signaling pathway proteins were detected in cells with tRF-27 overexpression (tRF-27 OE) and knockdown tRF-27 (tRF-27 IN) in a trastuzumab-stimulating environment. TRF-27 did not affect the proteins upstream MTORC1, but significantly promoted MTORC1 activation. C-F. Immunofluorescence with G3BP1,G3BP2, LAMP1, Tuberin and RPTOR. When tRF-27 was overexpressed, the colocalization of G3BP1 and Tuberin with LAMP1 was hindered. Knocking down tRF-27 increased the colocalization of G3BP1 and LAMP1. Overexpressing tRF-27 also separated G3BP1 from RPTOR. G. Overexpression of tRF-27 promoted phosphorylation of Tuberin.
Figure 8
Figure 8
Phosphorylation of Tuberin was associated with trastuzumab resistance. Tumor specimens from HER2-positive breast cancer patients who had received trastuzumab-containing regimens were divided into trastuzumab-sensitive and trastuzumab-resistant groups, based on whether the patients' Miller-Payne scores were greater than 2. A-B. Tissue samples from two additional patients who had not received neoadjuvant therapy were used as controls. Trastuzumab treatment did not affect G3BP1 expression, but trastuzumab-sensitive patients showed stronger expression of p-Tuberin and relatively weaker expression of p-4EBP1.
Figure 9
Figure 9
tRF-27 promoted the phosphorylation of Tuberin and the activation of MTORC1. A. Immunohistochemistry of mouse xenograft tumor specimens. B. As shown by the immunohistochemical results, overexpression of tRF-27(OE) inhibited the phosphorylation of Tuberin and promoted the activation of MTORC1. Inhibition of tRF-27(IN) promoted the phosphorylation of Tuberin and inhibited the activation of MTORC1. C. High levels of tRF-27 blocked lysosomal localization of TSC complexes by competitively combining G3BPs with LAMPs. As the inhibition on MTORC1 was relieved, the growth of trastuzumab-stimulated cells was promoted, thus enhancing the tolerance of tumor cells to trastuzumab.
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References

    1. Harbeck N, Gnant M. Breast cancer. Lancet. 2017;389:1134–50. - PubMed
    1. Leyland-Jones B. Trastuzumab: hopes and realities. The Lancet Oncology. 2002;3:137–44. - PubMed
    1. Valabrega G, Montemurro F, Aglietta M. Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol. 2007;18:977–84. - PubMed
    1. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med. 2000;6:443–6. - PubMed
    1. Butti R, Das S, Gunasekaran VP, Yadav AS, Kumar D, Kundu GC. Receptor tyrosine kinases (RTKs) in breast cancer: signaling, therapeutic implications and challenges. Mol Cancer. 2018;17:34. - PMC - PubMed