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Cancer: When Normal Stem Cell Function Goes Awry

 

pETS2 cancerAs we began to genetically dissect the underlying mechanisms by which skin stem cells transition from a non-tissue-generating to a tissue-generating mode, we realized that when we over-activated the pathway genetically in mice, they became more capable of repairing wounds but also more prone to skin cancers. Conversely, when we under-activated the pathway, mice repaired wounds more slowly but also became more resistant to cancers. These findings revealed that malignant progression derails the basic mechanisms that normal stem cells use to make and repair tissues. Indeed our recent chromatin landscaping studies have unearthed remarkable parallels between how stem cells survive as they exit their niche to repair a skin wound and the early steps in malignancy (Ge et al., 2017). As the cancer progresses tumor-initiating cells elevate chromatin accessibility through a process involving oncogenic RAS/MAPK modification of transcription factor ETS2 (Yang et al., 2015). Thus, cancer truly is a wound that never heals!

SCCs of the skin are the second most common cancer world-wide, and if neglected, can metastasize to the lung and other less common sites. More broadly, SCCs can occur in most stratified epithelial tissues, including the cervix, anogenital tissues, lung, esophagus and oral tissues of the head and neck. Head and neck cancers alone are the 6th most deadly cancer world-wide with a devastating 50% mortality rate. Given their aggressiveness and frequency, surprisingly little is known about these cancers, although they often arise from oncogenic mutations in the RAS/MAPK pathway, and their tumor-initiating cells are often typified by expression of CD34, α6 integrin, SOX9 and SOX2. Hundreds of additional changes distinguish the tumor-initiating SCC cells from epidermal and hair follicle stem cells, from which SCCs can arise (Schober et al., 2011).tumor

Using our powerful lentiviral delivery system, we’ve sifted through the complexity of the hundreds of gene differences revealed by our mouse SSCs and also the human cancer sequencing project. We’ve performed large scale genetic screens in vivo in mice to identify which of the myriads of gene changes are likely to reflect alterations in oncogenic, tumor-suppressor and/or microRNA gene expression and which stimulate proliferation versus which repress differentiation (Beronja et al., 2013; Schramek et al., 2014; Ge et al., 2016; Asare et al., 2017).

On another route, we were intrigued by the fact that two distinct populations of tumor-initiating cells could be purified from SCCs. They differed by their response to TGFβ, but we were unable to identify a hierarchical relation between the two populations (Schober et al., 2011). Probing deeper into mechanism, we devised a lentiviral vector that harbored two genes: 1) a constitutively active tetracycline-sensitive transactivator and 2) a TGFβ-sensitive gene expressing mCherry and a tamoxifen inducible CreER. When transduced into a tetracycline-inducible oncogenic HRas mutant X Rosa26fl-STOP-fl-YFP mouse embryo at low titer, we could add tetracycline to activate mutant Ras, but only in transduced skin clones, thereby controlling tumor burden. lineage-traced tumor regrowthAdditionally, as tumors developed and progressed to SCCs, we detected mCherry exclusively in the TGFβ-responding tumor-initiating stem cells, enabling us to show that these stem cells were ones adjacent to blood vessels. Moreover, when we added tamoxifen, we could lineage trace these SCs, showing that this population of stem cells responded to TGFβ by becoming slower-cycling but invasive, undergoing an EMT-like transition. Most intriguingly, these TGFβ-responding SCs resisted chemotherapy, and regrew the tumor (Oshimori et al., 2015).

In the past year, we’ve also interrogated the underlying basis for tumor relapse in SCCs treated with adoptive cell transfer (Car-T-like) immunotherapy (Miao et al., 2019). After treating mice with cytotoxic T cells that recognize an engineered surface antigen in our SCC model, 90% of the tumor cells were effectively attacked and eliminated. Eventually, the tumors relapsed. Our analyses revealed that surviving cells were once again the TGFβ-responding SCs. Seeking mechanism, we discovered that intriguingly, these tumor-initiating cells protect themselves by expressing low levels of CD80, a relative to PDL1 (Miao et al., 2019). These findings suggest that tumor-initiating cells possess an arsenal of weapons that enable them to protect themselves and survive.

CSC chartOverall, our findings show that heterogeneity in the tumor microenvironment can lead to striking changes in malignant stem cell behavior. As a blood vessel comes close to the tumor, it brings with it a milieu rich in TGFβ, prompting nearby stem cells to become slow-cycling, invasive and resistant to chemo and immunotherapy. More distant from the vessel, the stem cells are fast-cycling, giving rise to the bulk of the tumor. These SCs self-renew and generate many more tumor-initiating cells, and since blood vessels come and go, the process is dynamic, yielding a double edge sword to cancer, and providing an explanation for why cancers are so difficult to treat. That said, if our findings on mice hold for human SCCs, they offer a number of new insights which could be useful in informing new therapeutics and treatment regimens for these cancers.

 As we began to genetically dissect the underlying mechanisms by which skin stem cells transition from a non-tissue-generating to a tissue-generating mode, we realized that when we over-act