Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5⁻ and formed distant metastases in which Lgr5⁺ CSCs appeared. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5⁻ cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors.

Significance: Stem cells crucially depend on their complex microenvironment, also called niche. The niche is defined as an anatomic site, consisting of specialized niche cells. These niche cells anchor stem cells and provide the stem cells with physical protection and essential growth and maintenance signals. In the murine small intestinal crypts, Paneth cells constitute an important part of cellular niche for Lgr5 ⁺ stem cells with which they are intermingled. Paneth cells provide molecules such as Wnt3, EGF, and Notch ligands to maintain intestinal stem cell. There exists no typical Paneth cell in the colon. Here, we show that Reg4-expressing deep crypt secretory cells function as the colon equivalent of Paneth cells.

Significance: Metastasis is the main cause of cancer death, but the underlying mechanisms are largely unknown. Here, we developed an orthotopic organoid transplantation approach and used engineered human colon tumor organoids to study the contribution of common CRC mutations to metastasis. Using this approach, we show that the combination of oncogenic mutations in Wnt, EGFR, P53, and TGF-β signaling pathways facilitates efficient tumor cell migration and metastasis. These mutations allow growth independent of stem cell niche factors, enabling cells to grow at foreign distant sites that lack these factors. Our findings suggest that metastasis is a direct consequence of acquired niche independency.

Summary: The C subunit of the vacuolar H+-ATPase or V-ATPase regulates the activity and assembly of the proton pump at cellular membranes. It has been shown to be strongly upregulated in oral squamous cell carcinoma, a highly metastatic epithelial cancer. In addition, increased V-ATPase activity appears to correlate with invasiveness of cancer cells, but the underlying mechanism is largely unknown. Using the Drosophila wing imaginal epithelium as an in vivo model system, we demonstrate that overexpression of Vha44, the Drosophila orthologue of the C subunit, causes a tumor-like tissue transformation in cells of the wing epithelium. Overexpressing cells are excluded from the epithelium and acquire invasive properties while displaying high apoptotic rates. Blocking apoptosis in these cells unmasks a strong proliferation stimulus, leading to overgrowth. Furthermore, we show that excess Vha44 greatly increases acidification of endocytic compartments and interferes with endosomal trafficking. As a result, cargoes such as GFP-Lamp1 and Notch accumulate in highly acidified enlarged endolysosomal compartments. Consistent with previous reports on the endocytic activation of Eiger/JNK signaling, we find that V-ATPase stimulation by Vha44 causes JNK signaling activation whereas downmodulation of JNK signaling rescues the invasive phenotypes. In summary, our in vivo-findings demonstrate that increased levels of V-ATPase C subunit induce a Eiger/JNK-dependent cell transformation within an epithelial organ that recapitulates early carcinoma stages.