Human TNFRSF1A Knockout Cell Line-HeLa

Microtubule-targeting agents (MTAs) are one of the most widely used classes of chemotherapeutic drugs, and their mechanism of action has long been assumed to be blocking mitosis in rapidly dividing tumor cells. Contrary to this view, here researchers show that - in many cancer cell types - MTAs act by triggering membrane TNF (memTNF)-mediated cell-to-cell killing, which is very different from other non-MTA cell cycle blockers. Both memTNF and solTNF bind to TNF receptor 1 (TNFR1, also known as tumor necrosis factor receptor superfamily member 1A (TNFRSF1A)), thereby activating downstream cell death pathways. In cells that do not express RIP3 or have defective RIP3 signaling, TNFR1 activates the initiator protease caspase-8, which subsequently cleaves and activates the downstream executioner proteases caspase-3/7, leading to apoptosis. Here, researchers uncover a highly specific feature of the MTA family of drugs that distinguishes them from other mitotic blockers: both microtubule depolymerizers and stabilizers can directly induce TNF signaling-mediated tumor cell death, either by apoptosis in RIP3-deficient cancer cells or by necroptosis in RIP3-expressing cancer cells. Surprisingly, this MTA-induced cancer cell death was dependent on JNK/c-Jun-regulated accumulation of membrane TNF (memTNF) but not solTNF, through which it induces cancer cell killing by memTNF.

Most human cancer cells, such as HeLa cells, are resistant to necroptosis but sensitive to apoptosis due to the lack of expression of the necroptosis-essential gene RIP3. Here, the researchers tested the cell death response of human cancer cell lines lacking RIP3 to MTA. The study found that MTA also induced TNF-dependent apoptosis in HeLa cells. Blocking soluble TNF shedding by inhibiting TACE activity did not interfere with MTA-induced apoptosis; when TNFR1 was knocked out, cell death was abolished (Figure 1a-c). Ectopic expression of TNFR1 in TNFR1 (TNFRSF1A) knockout HeLa cells restored the apoptotic response to MTA (Figure 4d). The researchers next confirmed that MTA induced apoptosis in a variety of human cancer cell lines lacking RIP3 (Figure 1e-g). In fact, the mRNA levels of JUN and membrane-bound TNF in these human cancer cells were also upregulated by MTA treatment (Figure 1h,i).

Figure 1. MTAs induce memTNF-mediated apoptosis in RIP3-deficient human carcinoma cell lines. (Zhang, Jing, et al. 2020)