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, 22 (9), 1056-61

Loss of Cohesin Complex Components STAG2 or STAG3 Confers Resistance to BRAF Inhibition in Melanoma

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Loss of Cohesin Complex Components STAG2 or STAG3 Confers Resistance to BRAF Inhibition in Melanoma

Che-Hung Shen et al. Nat Med.

Abstract

The protein kinase B-Raf proto-oncogene, serine/threonine kinase (BRAF) is an oncogenic driver and therapeutic target in melanoma. Inhibitors of BRAF (BRAFi) have shown high response rates and extended survival in patients with melanoma who bear tumors that express mutations encoding BRAF proteins mutant at Val600, but a vast majority of these patients develop drug resistance. Here we show that loss of stromal antigen 2 (STAG2) or STAG3, which encode subunits of the cohesin complex, in melanoma cells results in resistance to BRAFi. We identified loss-of-function mutations in STAG2, as well as decreased expression of STAG2 or STAG3 proteins in several tumor samples from patients with acquired resistance to BRAFi and in BRAFi-resistant melanoma cell lines. Knockdown of STAG2 or STAG3 expression decreased sensitivity of BRAF(Val600Glu)-mutant melanoma cells and xenograft tumors to BRAFi. Loss of STAG2 inhibited CCCTC-binding-factor-mediated expression of dual specificity phosphatase 6 (DUSP6), leading to reactivation of mitogen-activated protein kinase (MAPK) signaling (via the MAPKs ERK1 and ERK2; hereafter referred to as ERK). Our studies unveil a previously unknown genetic mechanism of BRAFi resistance and provide new insights into the tumor suppressor function of STAG2 and STAG3.

Figures

Figure 1
Figure 1. Decreased expression of STAG2 and STAG3 in BRAFi-resistant melanoma primary patient tumors and cell lines
(a) Sanger sequencing analysis of the STAG2 locus in pre-treatment (left) and post-relapse (right) biopsies from a patient with recurrent disease following vemurafenib treatment. (b) Expression of cohesion complex components, STAG1, STAG2, STAG3 and RAD21 in a panel of melanoma BRAFi-resistant cell lines and their parental BRAFi-sensitive counterparts was measured by western blotting. GAPDH was used a loading control. P: parental; BR: BRAFi resistant. BMR: BRAFi and MEKi double resistant. (c) Immunohistochemical analyses of STAG2 and STAG3 in pairs of pre-treatment (left) and post-relapse (right) tumor samples from patients treated with BRAFi monotherapy or BRAFi and MEKi combination therapy. Two representative patients are shown for STAG2 and for STAG3. Scale bar: 50 microns.
Figure 2
Figure 2. Knockdown of STAG2 or STAG3 decreases BRAFi sensitivity in BRAF mutant melanoma cells
(a) Viability of A375 cells after treatment with varying concentrations of dabrafenib for 3 d. Experiment was performed 3 times. Data are mean ± s.e.m. (b) A375 cells were treated with dabrafenib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (c) Viability of SKMEL28 cells after treatment with varying concentrations of vemurafenib for 3 d. Experiment was performed 3 times. Data are mean ± s.e.m. (d) SKMEL28 cells were treated with vemurafenib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (e) SKMEL30 cells were treated with trametinib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (f) SKMEL30 cells were treated with trametinib as indicated in clonogenic growth assays. Experiment was performed 3 times. Scale bar: 5 mm. (g) Viability of A375 cells after treatment with varying concentrations of dabrafenib for 3 d. Experiment was performed 3 times. Data are mean ± s.e.m. (h) A375 cells were treated with vemurafenib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (i) WM902-BR cells stably expressing control vector, FLAG-tagged wild-type STAG2 (WT), Lys1083* (K*) or Asp193Asn (DN) mutants were treated with 3 μM vemurafenib for 2 h. Cell lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (j) WM902-BR cells were used in soft agar assays in the presence or absence of 3 μM vemurafenib. Experiment was performed 3 times. Scale bar: 5 mm.
Figure 3
Figure 3. Knockdown of STAG2 or STAG3 impairs the effects of vemurafenib on inhibiting melanoma xenograft tumor growth in vivo
(a) Nude mice bearing xenograft tumors of A375 cells stably expressing pTRIPZ-shSTAG2#60 were treated with vehicle, doxycyline (Dox), vemurafenib (Vem), or both doxycyline and vemurafenib. Unpaired two-tailed Student's t-test was performed to compare between two groups of mice that were treated with vemurafenib (mean ± s.e.m. * P < 0.05). The data variance is similar between groups. n = 5–7. (b) Waterfall plots showing the percent change in tumor volume at day 7 for the individual tumors in each treatment group of the STAG2 knockdown experiment. (c) Representative images of mouse tumor samples from the STAG2 knockdown experiment subjected for various immunohistochemical analyses as indicated. Scale bar: 50 μm. (d) Nude mice bearing xenograft tumors of A375 cells stably expressing pLKO-shSTAG3#69 were treated with control or vemurafenib (Vem) diet. Unpaired two-tailed Student's t-test was performed to compare between two groups of mice that were treated with vemurafenib (mean ± s.e.m. * P < 0.05). The data variance is similar between groups. n = 5–7. (e) Waterfall plots showing the percent change in tumor volume at day 8 for the individual tumors in each treatment group of the STAG3 knockdown experiment. (f) Representative images of mouse tumor samples from the STAG3 knockdown experiment subjected for various immunohistochemical analyses as indicated. Scale bar: 50 μm.
Figure 4
Figure 4. STAG2 regulates ERK activity through controlling expression of DUSP6
(a) Total RNA from A375 cells were isolated, reverse transcribed, and expression levels of DUSP4 and DUSP6 were analyzed by qPCR. mRNA levels were calculated relative to the scrambled control with housekeeping GAPDH gene as reference. n = 3 biological replicates. Data are mean ± s.e.m. The P values were determined using two-tailed Student's t-test, **** P < 0.0001. The data variance is similar between groups. (b) M14 or A375 cells expressing STAG2 inducible shRNA were cultured in the presence or absence of doxycycline for 5 d before lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (c) Lysates from M14 or A375 cells expressing STAG3 shRNA#71 or scrambled control were used for western blotting with indicated antibodies. Experiment was performed 3 times. (d) Lysates from HEK293 cells transfected with indicated constructs were used for western blotting with indicated antibodies. K*, Lys1083*; DN, Asp193Asn. Experiment was performed 3 times. (e) Genomic structure of DUSP6 gene showing the locations of amplified regions by ChIP-qPCR. R1: CTCF binding region, R2: non-specific region. (f) A375 cells expressing STAG2 inducible shRNA pTRIPZ-shSTAG2#60 were cultured in the presence or absence of doxycycline for 5 d before ChIP-qPCR assays were performed. Chromatins were immuoprecipitated using CTCF antibody or rabbit IgG. IP-ed chromatins were examined using qPCR with primers for R1 and R2 regions of DUSP6 and H19. Results are expressed as fold enrichment relative to the non-specific region (R2). n = 3 biological replicates. Data are mean ± s.e.m. The P values were determined using two-tailed Student's t-test, * P < 0.05. The data variance is similar between groups. (g) Chromatins of LOX-IVMI cells stable expressing FLAG-tagged wild-type STAG2 (WT), Lys1083* (K*) or Asp193Asn (DN) mutants were immuoprecipitated using CTCF antibody or rabbit IgG. n = 3 biological replicates. Data are mean ± s.e.m. The P values were determined using two-tailed Student's t-test, ** P < 0.01; **** P < 0.0001. The data variance is similar between groups. (h) A375 cells expressing STAG2 inducible shRNA pTRIPZ-shSTAG2#60 were infected with lentivirus expressing MYC-DUSP6 or control vector. Cells were cultured in the presence or absence of doxycycline for 5 d and treated with 0.3 μM vemurafenib for 2h before lysates were used for western blotting with indicated antibodies. Experiment was performed 3 times. (i) A375 cells expressing STAG2 inducible shRNA pTRIPZ-shSTAG2#60 together MYC-tagged DUSP6 or control vector were treated with vemurafenib as indicated in clonogenic growth assays. Experiment was performed 3 times. Scale bar: 5 mm.

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