Molecular Medicine Israel

On the origin of metastases: Induction of pro-metastatic states after impending cell death via ER stress, reprogramming, and a cytokine storm


  • Tumor cells that survive impending death become stable pro-metastatic tumor cells, PAMEs
  • PAMEs display molecularly defined pro-metastatic states and form distant metastases
  • ER stress PERK-CHOP, GLI, and NANOG underlie the induction of PAMEs
  • A PAME cytokine storm induces PIMs from nearby tumor cells, which help PAMEs metastasize


How metastatic cells arise is unclear. Here, we search for the induction of recently characterized pro-metastatic states as a surrogate for the origin of metastasis. Since cell-death-inducing therapies can paradoxically promote metastasis, we ask if such treatments induce pro-metastatic states in human colon cancer cells. We find that post-near-death cells acquire pro-metastatic states (PAMEs) and form distant metastases in vivo. These PAME (“let’s go” in Greek) cells exhibit a multifactorial cytokine storm as well as signs of enhanced endoplasmic reticulum (ER) stress and nuclear reprogramming, requiring CXCL8, INSL4, IL32, PERK-CHOP, and NANOG. PAMEs induce neighboring tumor cells to become PAME-induced migratory cells (PIMs): highly migratory cells that re-enact the storm and enhance PAME migration. Metastases are thus proposed to originate from the induction of pro-metastatic states through intrinsic and extrinsic cues in a pro-metastatic tumoral ecosystem, driven by an impending cell-death experience involving ER stress modulation, metastatic reprogramming, and paracrine recruitment via a cytokine storm.


Multiple mechanisms underlie the dissemination, homing, and distant seeding of metastatic cells, which cause the bulk of cancer-related deaths (Mehlen and Puisieux, 2006Lambert et al., 2017Wortzel et al., 2019Lu and Kang, 2019Hu et al., 2019Follain et al., 2020Massagué and Ganesh, 2021Bertocchi et al., 2021). The origin of metastasis, however, remains obscure. Cells that give rise to metastases must first acquire pro-metastatic states inside the primary tumor before engaging the migratory machinery to eventually form secondary tumor masses elsewhere. We recently described a small cell group, the METSP, with pro-metastatic states in a heterogeneous, primary human colon cancer population using spiked-single-cell RNA sequencing (scRNA-seq) (Bernal et al., 2020). This and a previous study (Mishra et al., 2019) identified critical intra-tumoral cell interactions that modulate the acquisition of metastatic properties, consistent with the involvement of signaling and cell plasticity (Fidler and Kripke, 1977Miller, 1983Varnat et al., 2010Singovski et al., 2016). However, how METSP states appear in the first place is unknown.Anti-cancer therapies with cell-death-inducing treatments (Longley et al., 2003Comella et al., 2009Carneiro and El-Deiry, 2020) can paradoxically enhance metastasis (Imamura et al., 1990Wild-Bode et al., 2001Poth et al., 2010Vyas et al., 2014Liu et al., 2015Middleton et al., 2018Karagiannis et al., 2019). How this comes about is not clear, although stemness acquisition, anastasis, senescence, and changes in the microenvironment have all been proposed to contribute (Tang et al., 2012Tang et al., 2017Ding et al., 2016Tato-Costa et al., 2016Sun et al., 2017Sun et al., 2020Xu et al., 2018Middleton et al., 2018Guillon et al., 2019Berthenet et al., 2020). Cells surviving acute drug-induced apoptosis can display oncogenic traits including epithelial-to-mesenchymal transition (EMT), the modulation of epigenetic remodelers, and limited migration (Tang et al., 2012Ichim et al., 2015Tato-Costa et al., 2016Sun et al., 2017Seervi et al., 2019Berthenet et al., 2020). However, as many of these studies selected cells from populations treated with apoptosis-inducing drugs at sub-lethal levels (Tang et al., 2012Sun et al., 2017Berthenet et al., 2020), it remained unclear if the used cells were actually fated to die, and thus it is unknown how phenotypes may match cell states.Survival from late apoptosis commonly triggered by the kinase inhibitor staurosporine (STS) (Wang et al., 2015aWang et al., 2015bXu et al., 2018) can be obtained through pharmacological inhibition of CASPASE activity with Q-VD-OPh and of mitochondrial outer-membrane permeabilization through the voltage-dependent anion channel blocker DIDS (Caserta et al., 2003Liu et al., 2008). Cells obtained in this manner have been utilized to address regenerative processes. For instance, apoptosis-surviving myotubes dedifferentiate, reprogram to acquire progenitor cell properties, and participate in the regeneration of limbs and muscle (Wang et al., 2015aWang et al., 2015bBulatovic et al., 2015). Using this approach here, we ask if METSP states, which show enriched expression of the endogenous CASPASE inhibitors BIRC3 and XIAP (Bernal et al., 2020), can be induced in primary tumor cells that escape impending total population (apocalyptic) apoptotic death, thus representing an origin of metastases.


 PAME induction through rescue from impending death

Cell-lethal STS treatments followed by their blockade 6 h later with the apoptotic inhibitors Q-VD-OPh plus DIDS (BLOCK) (Wang et al., 2015aWang et al., 2015bFigures 1A and 1B ) were performed on heterogeneous colon cancer CC14 cells (Varnat et al., 2009). Cells only treated with DMSO or BLOCK (control) were unaffected (Figures 1B and S1C), whereas 100% of the cells treated with STS died with apoptotic phenotypes (Figures 1B, 1C, S1A, and S1B), providing a clean background. Rescued PAMEs, obtained by STS + BLOCK with recovery of 10% after 5 days, proliferated and incorporated 5-ethynyl-2′-deoxyuridine (EdU) (Figures 1C, S1C, and S1D).

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