How activating the protective and cytotoxic anticipatory unfolded protein response controls transcription and cancer cell death

When bound to its ligand estrogen, estrogen receptor (ER) dimerizes and regulates target genes via directly binding to estrogen response element (ERE) or by interacting with other transcription factors which have binding sites located in or near the promoter of the target genes. In addition to its classical genomic action, E2, acting through ER⍺, can activate the anticipatory unfolded protein response (a-UPR) in the absence of endoplasmic reticulum stress and prior to the accumulation of misfolded/unfolded protein. Activation of the a-UPR is a recently described action for E2:ER⍺ that can protect breast cancer cells and contributes to antiestrogen resistance in ER⍺ breast cancer. Here, we describe how the rapid activation of the a-UPR modulates the subsequent activation of the E2:ER⍺ genomic program via calcium (Ca2+) signaling. The intracellular Ca2+ increase resulting from the activation of a-UPR enhances ER⍺ nuclear translocation and modulates ER⍺:DNA binding near genes related to cell proliferation, hypoxic stress response, and cell migration. The data suggests that the sensor system that recognizes increased intracellular Ca2+ and couples to transcription is based on activation of the Ca2+ binding protein calmodulin (CaM) and calcium/calmodulin-dependent kinase 2 (CaMK2), leading to the activation of cyclin dependent kinase 7 (CDK7). Furthermore, we discovered Ca2+ signaling from the a-UPR activation is essential in ligand-dependent phosphorylation of ER⍺ at serine 118 (Ser118) via the Ca2+:CaM-CaMK2-CDK7 pathway. Notably, genes sensitive to intracellular calcium increase are phosphor-ER⍺Ser118 dependent in HeLaER⍺ cells, providing additional evidence that calcium is modulating a subset of E2:ER⍺-regulated genes via Ca2+ signal-regulated ER⍺ phosphorylation. Recently, our lab identified a new small molecule that hyperactivates the a-UPR via ER⍺ to target and kill cancer cells. The second generation of small molecule ER⍺ biomodulator, ErSO, showed a promise in killing ER⍺-positive endometrial cancer cells. Upon binding ER⍺, ErSO elicits a rapid increase in cytosol calcium by opening IP3R channels located in the endoplasmic reticulum membrane. We identified that this calcium signal is essential in the ErSO-induced cell death pathway in endometrial cancer cells. The increase in calcium concentration in the cytosol activates the transient receptor potential cation channel subfamily M member 4 (TRPM4) in the plasma membrane, causing an influx of sodium ions from the extracellular space into the cytosol. In response to a sudden change in the cytosolic sodium ion concentration, chloride ion enters to balance the charge, and water rushes into the cytosol to maintain osmolarity. The disruption in osmotic balance and ion homeostasis lead to extensive cell swelling which elicits strong and sustained activation of the UPR and in some cases membrane rupture and necrotic cell death. Following TRPM4 activation, endometrial cancer cells try to restore the disrupted ion homeostasis across the plasma membrane by pumping out sodium ions via the Na+/K+ ATPase. However, due to the sustained sodium influx through TRPM4 opening, continuous Na/K ATPase activity creates a futile cycle leading to significant ATP depletion, leading to cell death. Cell swelling, ATP depletion, and loss of membrane integrity suggest ErSO kills endometrial cancer cells via necrotic cell death and is downstream of calcium release from the endoplasmic reticulum resulted from activation of the a-UPR through ER⍺.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste