Abstract
Cellular dormancy and heterogeneity in cell cycle length provide important explanations for treatment failure after adjuvant therapy with S-phase cytotoxics in colorectal cancer (CRC), yet the molecular control of the dormant versus cycling state remains unknown. We sought to understand the molecular features of dormant CRC cells to facilitate rationale identification of compounds to target both dormant and cycling tumor cells. Unexpectedly, we demonstrate that dormant CRC cells are differentiated, yet retain clonogenic capacity. Mouse organoid drug screening identifies that itraconazole generates spheroid collapse and loss of dormancy. Human CRC cell dormancy and tumor growth can also be perturbed by itraconazole, which is found to inhibit Wnt signaling through noncanonical hedgehog signaling. Preclinical validation shows itraconazole to be effective in multiple assays through Wnt inhibition, causing both cycling and dormant cells to switch to global senescence. These data provide preclinical evidence to support an early phase trial of itraconazole in CRC.
Introduction
Colorectal cancer (CRC) is the third most common cancer in the Western World. CRC is a heterogeneous disease and recent large scale molecular studies have identified clinically relevant overlapping subgroups that can be identified in primary tumors, primary cultures, xenografts, and traditional cell lines (De Sousa E Melo et al., 2013; Guinney et al., 2015; Linnekamp et al., 2018). This intertumoral heterogeneity is a major explanation for differential chemotherapy responses and clinical progression. Although recent advances in oncological treatment have generated marked improvements for patients with CRC, many who receive adjuvant therapy ultimately die as a result of relapse with systemic disease. There are several explanations for tumor recurrence, including cellular dormancy or quiescence that allow cancer cells to persist and reenter the cell cycle after a latent period or therapy-induced stimulation. Across cancer types, cellular dormancy has been shown to represent an important hallmark of cancer cells that facilitates immune evasion and avoidance of targeted death by S-phase cytotoxics (Kreso et al., 2013; Malladi et al., 2016). From a functional perspective, dormant CRC cells have been found to be rare, chemoresistant, and yet highly clonogenic, features compatible with a stem cell–like phenotype (Moore et al., 2012; Kreso et al., 2013). However, their true molecular identity and the mechanisms underlying dormancy remain elusive, and there is an urgent need to identify compounds that can perturb this dormant state to enable more complete cancer cell killing to prevent late recurrence.
In the normal intestine there are two stem cell populations: one rapidly dividing and another quiescent reserve population that becomes activated during tissue injury (Clevers, 2013). It is increasingly recognized that premalignant adenomas and malignant tumors contain many similar cell types as that found in the tissue of origin (Verga Falzacappa et al., 2012). Two very recent studies have identified and characterized cancer stem cell (CSC) populations in CRC (De Sousa E Melo et al., 2017; Shimokawa et al., 2017). In one study, De Sousa E Melo et al. demonstrate that liver metastases arising from primary colon cancers are highly dependent on Lgr5-expressing CSCs, and elimination of these cells dramatically decreases both the formation and maintenance of liver metastases, whereas in primary tumors they are redundant (De Sousa E Melo et al., 2017). In an alternative study, Shimokawa et al. (2017) show two distinct cell populations exist in human CRCs: a differentiated cell population with limited self-renewal capacity expressing Keratin 20 (Krt20) and a proliferative CSC population expressing Lgr5. Intriguingly, targeting of Lgr5+ CSCs leads to dedifferentiation of the Krt20 population to an Lgr5+ status, sustaining continued tumor growth. Whether both populations are present throughout all molecular subtypes of CRC and what signaling pathways control these divergent behaviors is unknown. However, it is clear, to achieve therapeutic benefit, Lgr5+ CSCs and less proliferative Krt20+ cells must both be targeted.
The Wnt pathway is commonly hyperactivated in CRC and plays a pivotal role in intestinal stem cell maintenance. High Wnt activity has been shown to also define CSCs (Vermeulen et al., 2010). Although much is now understood about how Wnt signaling functions and several experimental compounds have been tested, it has been impossible to develop a clinically useful drug that can target the pathway. In addition to Wnt, there are several other autocrine- and paracrine-signaling pathways that are involved in controlling intestinal stem cell behavior/kinetics during epithelial homeostasis and regeneration (Beumer and Clevers, 2016). Further, there is significant cross talk between many of these signaling pathways. Perturbation of individual pathways has been shown to alter the behavior of stem cells, progenitor populations, and the proportions of differentiated cells generated. Here, we seek to characterize dormant cells in different CRC subtypes to identify controlling pathways. We hypothesize that disrupting these pathways could cause departure from dormancy with subsequent proliferation, thus rendering the tumor as a whole more susceptible to conventional therapies that target dividing cells.
