Abstract
Outgrowth of metastases expressing ERα mutations Y537S and D538G is common after endocrine therapy for estrogen receptor α (ERα) positive breast cancer. The effect of replacing wild type ERα in breast cancer cells with these mutations was unclear. We used the CRISPR-Cas9 genome editing system and homology directed repair to isolate and characterize 14 T47D cell lines in which ERαY537S or ERαD538G replace one or both wild-type ERα genes. In 2-dimensional, and in quantitative anchorage-independent 3-dimensional cell culture, ERαY537S and ERαD538G cells exhibited estrogen-independent growth. A progestin further increased their already substantial proliferation in micromolar 4-hydroxytamoxifen and fulvestrant/ICI 182,780 (ICI). Our recently described ERα biomodulator, BHPI, which hyperactivates the unfolded protein response (UPR), completely blocked proliferation. In ERαY537S and ERαD538G cells, estrogen-ERα target genes were constitutively active and partially antiestrogen resistant. The UPR marker sp-XBP1 was constitutively activated in ERαY537S cells and further induced by progesterone in both cell lines. UPR-regulated genes associated with tamoxifen resistance, including the oncogenic chaperone BiP/GRP78, were upregulated. ICI displayed a greater than 2 fold reduction in its ability to induce ERαY537S and ERαD538G degradation. Progestins, UPR activation and perhaps reduced ICI-stimulated ERα degradation likely contribute to antiestrogen resistance seen in ERαY537S and ERαD538G cells.
Highlights
The most common application of the CRISPR-Cas[9] system is targeted gene inactivation by non homologous end joining (NHEJ) to repair the Cas[9] generated DNA break, when a homologous repair donor is present, a homology-directed repair process (HDR) can precisely insert a sequence containing the desired modification into the gene of interest
Previous transfection studies suggested ERαY537S and ERαD538G cells grow without E21–3
While our use of 2 guide sequences to increase the frequency of HDR may increase the possibility of off-target events[42,43,44], our ability to confirm key observations in 4 ERαY537S and 3 ERαD538G clonal cell lines strongly suggests that the observed properties are due to ERαgene replacement
Summary
The most common application of the CRISPR-Cas[9] system is targeted gene inactivation by non homologous end joining (NHEJ) to repair the Cas[9] generated DNA break, when a homologous repair donor is present, a homology-directed repair process (HDR) can precisely insert a sequence containing the desired modification into the gene of interest. We used the CRISPR-Cas[9] gene editing system to generate 50 clonal cell lines with one or both copies of endogenous wild-type ERαreplaced with ERαY537S or ERαD538G. Activation of a UPR gene index at diagnosis is a powerful prognostic indicator, tightly correlated with subsequent resistance to tamoxifen therapy[20]. This ERα-regulated UPR pathway is targeted by BHPI, our recently described noncompetitive ERαbiomodulator. We describe the effects of OHT, ICI and BHPI on proliferation of the ERαY 537S and ERαD 538G cells in anchorage dependent and anchorage independent culture with and without a progestin, analyze gene expression, and evaluate estrogen-independent and progestin-stimulated UPR activation and reduced ERαdegradation as potential contributors to antiestrogen resistance
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