Ferraiuolo RM et al. (2017), The cyclin-like protein, SPY1, regulates the ER...

XB-ART-53636

Oncotarget 2017 Apr 04;814:23337-23352. doi: 10.18632/oncotarget.15578.

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The cyclin-like protein, SPY1, regulates the ERα and ERK1/2 pathways promoting tamoxifen resistance.

Ferraiuolo RM , Tubman J , Sinha I , Hamm C , Porter LA .

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The Ras/Raf/MEK/ERK pathway conveys growth factor and mitogen signalling to control the phosphorylation of a plethora of substrates regulating proliferation, survival, and migration. The Ras signalling pathway is frequently associated with poor prognosis and drug resistance in various cancers including those of the blood, breast and prostate. Activation of the downstream effector ERK does not always occur via a linear cascade of events; complicating the targeting of this pathway therapeutically. This work describes a novel positive feedback loop where the cell cycle regulatory factor Spy1 (RINGO; gene SPDYA) activates ERK1/2 in a MEK-independent fashion. Spy1 was originally isolated for the ability to stimulate Xenopus oocyte maturation via a MAPK-signalling pathway and is known to override apoptosis triggered by the DNA damage response. We demonstrate that mammalian Spy1-mediated ERK activation increases ligand-independent phosphorylation and activation of estrogen receptor α, correlating with a decrease in tamoxifen sensitivity. This could define a novel druggable mechanism driving proliferation and resistance in select cancers.

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Genes referenced: mapk1 ripk2 spdya znrd2

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	Figure 1. Spy1 is upregulated downstream of the estrogen receptor(A) MCF7 cells were treated with 50 nM of estradiol (E2) or vehicle control (DMSO) over the indicated time course. Representative blot (left), densitometry averages Spy1 and S118 (right). (B–C) Hek-293 cells were transfected with pEGFP-C1-ERα. B-Representative blot confirming expression. C-Treatment with 50 nM of E2 over the indicated time course. Representative blot (left), densitometry averages for Spy1 (right). (D) MCF7 cells infected with control, Spy1, or Cyclin E1.(E) Cells were infected pLKO, 2 constructs of shSpy1.1, shSpy1.2, shCyclin E, or rescue vectors, followed by SDS-PAGE and IB. Representative blot (left), densitometry averages for relative ER-S118 (right). (F) Representative blot (upper panel) showing phosphorylation status of ERK1 and ERK2 using phosphor-specific antibodies for pERK threonine or tyrosine sites. Lower panel depicts quantification of both sites for either ERK1 (left) or ERK2 (right). Error bars reflect SE between at least 3 separate experiments. Student's t-test was performed; p < 0.05, p < 0.01, **p < 0.001.
	Figure 2. Spy1-mediated ERK phosphorylation is CDK dependent(A–C) Hek-293 cells were transfected with the indicated constructs (along top of each representative blot and X-axis of each graph, including the empty vector control (pCS3). (A) Representative blot (left). Densitometry for Spy1 or pERK over multiple experiments (right). (B) Trypan blue exclusion assay was performed after 24 hours of incubation, total cell numbers presented. (C) Representative blot (left). Densitometry (right) as represented on Y-axis. Error bars reflect SE between at least 3 experiments. Student's t-test was performed; p < 0.05, p < 0.002, **p < 0.001.
	Figure 3. Spy1-mediated ERK phosphorylation is MEK-independent(A–C) Hek-293 cells were infected (A) or transfected (B and C) with the indicated constructs (along top of each representative blot and X-axis of each graph. (A) Cells were treated with 10 μM U0126 or vehicle control (DMSO). (A–C) Representative blot (left). Densitometry (right) as represented on Y-axis or text box. Error bars reflect SE between at least 3 experiments. Student's t-test was performed; p < 0.05, p < 0.002, **p < 0.001.
	Figure 4. Spy1 activation of ERK1/2 is dependent on Ras and RafHek-293 cells were transfected (A) or infected (B–D) with constructs indicated at the top of each blot (left). (A and C) were treated with inhibitors as indicated on the panels. (A, C–D) Densitometry of relative protein levels conducted over all experiments (right). Lower right panel (C) fold change ratio of pERK:Spy1 protein levels. Error bars reflect SE between at least 3 experiments. Student's t-test was performed;p < 0.05,p < 0.01, **p < 0.001.
	Figure 5. Spy1 levels affect tamoxifen response in vivo(A) MCF7 cells were infected with indicated constructs (along top of representative blot and X-axis of graph). Trypan blue exclusion assay was performed over indicated time course in the presence or absence of tamoxifen. Error bars reflect SE between triplicate experiments. (B) Representative images of injected zebrafish larvae expressing either empty control vector (top panel) or Spy1 overexpression vector (bottom panel) before (0 hpt) and after (24 hpt) treatment with either DMSO or 10 μM tamoxifen. The same fish is depicted at 0 and 24 hpt for each condition. Scale bar = 200 μm. Graph representing the mean fold change in foci, as quantified by fluorescence as compared to 0 hpt. n = 28–46 fish/treatment (excluding mortalities). Student′s t-test was performed; ns = not significant, p < 0.01, **p < 0.001. Scale bar = 200 μm.
	Figure 6. Spy1 levels regulate the response to tamoxifenMCF7 cells were infected with constructs indicated at the top of each representative blot and X-axis of each densitometry graph. Drug treatments are indicated in each panel. Tamoxifen (100 nM), MEK1/2 inhibitor (10 μM), ERK inhibitor (10 μM) and control MEK/ERK inhibitors (10 μM). (A) Representative blot (left) of pS118 without tamoxifen treatment. (B) Representative blot (left) and quantification of pS118 (right). (C) Representative blot (left) and quantification of pS118 (right). (D) Viable cell numbers after treatment assayed using trypan blue exclusion. Error bars reflect SE between at least 3 individual experiments. Student's t-test was performed; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001.
	Figure 7. Schematic diagram of proposed pathwayOur data supports that elevated levels of Spy1 can act upstream to activate ERK1/2 through a MEK-independent pathway. Our data supports that this depends on a direct interaction with p27, as well as Ras and Raf activation. A novel pathway has been described demonstrating a Raf downstream kinase, RIPK2 [55] capable of activating ERK1/2 in a MEK-independent fashion. Our data shows a dependence of Spy1-mediated effects on RIPK2. Spy1-mediated ERK1/2 activation can phosphorylate the ER on S118 and alter sensitivity to tamoxifen.

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	Figure 1. Spy1 is upregulated downstream of the estrogen receptor(A) MCF7 cells were treated with 50 nM of estradiol (E2) or vehicle control (DMSO) over the indicated time course. Representative blot (left), densitometry averages Spy1 and S118 (right). (B–C) Hek-293 cells were transfected with pEGFP-C1-ERα. B-Representative blot confirming expression. C-Treatment with 50 nM of E2 over the indicated time course. Representative blot (left), densitometry averages for Spy1 (right). (D) MCF7 cells infected with control, Spy1, or Cyclin E1.(E) Cells were infected pLKO, 2 constructs of shSpy1.1, shSpy1.2, shCyclin E, or rescue vectors, followed by SDS-PAGE and IB. Representative blot (left), densitometry averages for relative ER-S118 (right). (F) Representative blot (upper panel) showing phosphorylation status of ERK1 and ERK2 using phosphor-specific antibodies for pERK threonine or tyrosine sites. Lower panel depicts quantification of both sites for either ERK1 (left) or ERK2 (right). Error bars reflect SE between at least 3 separate experiments. Student's t-test was performed; p < 0.05, p < 0.01, **p < 0.001.
	Figure 2. Spy1-mediated ERK phosphorylation is CDK dependent(A–C) Hek-293 cells were transfected with the indicated constructs (along top of each representative blot and X-axis of each graph, including the empty vector control (pCS3). (A) Representative blot (left). Densitometry for Spy1 or pERK over multiple experiments (right). (B) Trypan blue exclusion assay was performed after 24 hours of incubation, total cell numbers presented. (C) Representative blot (left). Densitometry (right) as represented on Y-axis. Error bars reflect SE between at least 3 experiments. Student's t-test was performed; p < 0.05, p < 0.002, **p < 0.001.
	Figure 3. Spy1-mediated ERK phosphorylation is MEK-independent(A–C) Hek-293 cells were infected (A) or transfected (B and C) with the indicated constructs (along top of each representative blot and X-axis of each graph. (A) Cells were treated with 10 μM U0126 or vehicle control (DMSO). (A–C) Representative blot (left). Densitometry (right) as represented on Y-axis or text box. Error bars reflect SE between at least 3 experiments. Student's t-test was performed; p < 0.05, p < 0.002, **p < 0.001.
	Figure 4. Spy1 activation of ERK1/2 is dependent on Ras and RafHek-293 cells were transfected (A) or infected (B–D) with constructs indicated at the top of each blot (left). (A and C) were treated with inhibitors as indicated on the panels. (A, C–D) Densitometry of relative protein levels conducted over all experiments (right). Lower right panel (C) fold change ratio of pERK:Spy1 protein levels. Error bars reflect SE between at least 3 experiments. Student's t-test was performed;p < 0.05,p < 0.01, **p < 0.001.
	Figure 5. Spy1 levels affect tamoxifen response in vivo(A) MCF7 cells were infected with indicated constructs (along top of representative blot and X-axis of graph). Trypan blue exclusion assay was performed over indicated time course in the presence or absence of tamoxifen. Error bars reflect SE between triplicate experiments. (B) Representative images of injected zebrafish larvae expressing either empty control vector (top panel) or Spy1 overexpression vector (bottom panel) before (0 hpt) and after (24 hpt) treatment with either DMSO or 10 μM tamoxifen. The same fish is depicted at 0 and 24 hpt for each condition. Scale bar = 200 μm. Graph representing the mean fold change in foci, as quantified by fluorescence as compared to 0 hpt. n = 28–46 fish/treatment (excluding mortalities). Student′s t-test was performed; ns = not significant, p < 0.01, **p < 0.001. Scale bar = 200 μm.
	Figure 6. Spy1 levels regulate the response to tamoxifenMCF7 cells were infected with constructs indicated at the top of each representative blot and X-axis of each densitometry graph. Drug treatments are indicated in each panel. Tamoxifen (100 nM), MEK1/2 inhibitor (10 μM), ERK inhibitor (10 μM) and control MEK/ERK inhibitors (10 μM). (A) Representative blot (left) of pS118 without tamoxifen treatment. (B) Representative blot (left) and quantification of pS118 (right). (C) Representative blot (left) and quantification of pS118 (right). (D) Viable cell numbers after treatment assayed using trypan blue exclusion. Error bars reflect SE between at least 3 individual experiments. Student's t-test was performed; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001.
	Figure 7. Schematic diagram of proposed pathwayOur data supports that elevated levels of Spy1 can act upstream to activate ERK1/2 through a MEK-independent pathway. Our data supports that this depends on a direct interaction with p27, as well as Ras and Raf activation. A novel pathway has been described demonstrating a Raf downstream kinase, RIPK2 [55] capable of activating ERK1/2 in a MEK-independent fashion. Our data shows a dependence of Spy1-mediated effects on RIPK2. Spy1-mediated ERK1/2 activation can phosphorylate the ER on S118 and alter sensitivity to tamoxifen.