Pauliina Kallio

Prox1 promotes expansion of the colorectal cancer stem cell population to fuel tumor growth and ischemia resistance.

Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of these cells. We show here that a subpopulation of Prox1-transcription-factor-expressing cells have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth via induction of annexin A1 and reduction of the actin-binding protein filamin A, which has been implicated as a prognostic marker in CRC. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut.

Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-β

Significance The TGF-β/Smad pathway is mutated in the majority of late-stage colorectal cancers (CRCs), but its role in intestinal adenomas is unclear. We show here that intestinal epithelial cells, including the Lgr5+ stem cells, are sensitive to the TGF-β–induced apoptosis in adenomas and that this is mediated by the BH3-only protein Bim. Furthermore, the tumor-initiating Apc mutation increases, whereas the KRas oncogene decreases the TGF-β sensitivity. Our results provide important mechanistic insight into how TGF-β regulates intestinal adenoma development and show that drugs mimicking the effects of BH3-only proteins can induce apoptosis also in CRC cells that are resistant to TGF-β. In the majority of microsatellite-stable colorectal cancers (CRCs), an initiating mutation occurs in the adenomatous polyposis coli (APC) or β-catenin gene, activating the β-catenin/TCF pathway. The progression of resulting adenomas is associated with oncogenic activation of KRas and inactivation of the p53 and TGF-β/Smad functions. Most established CRC cell lines contain mutations in the TGF-β/Smad pathway, but little is known about the function of TGF-β in the early phases of intestinal tumorigenesis. We used mouse and human ex vivo 3D intestinal organoid cultures and in vivo mouse models to study the effect of TGF-β on the Lgr5+ intestinal stem cells and their progeny in intestinal adenomas. We found that the TGF-β–induced apoptosis in Apc-mutant organoids, including the Lgr5+ stem cells, was mediated by up-regulation of the BH3-only proapoptotic protein Bcl-2–like protein 11 (Bim). BH3-mimetic compounds recapitulated the effect of Bim not only in the adenomas but also in human CRC organoids that had lost responsiveness to TGF-β–induced apoptosis. However, wild-type intestinal crypts were markedly less sensitive to TGF-β than Apc-mutant adenomas, whereas the KRas oncogene increased resistance to TGF-β via the activation of the Erk1/2 kinase pathway, leading to Bim down-regulation. Our studies identify Bim as a critical mediator of TGF-β–induced apoptosis in intestinal adenomas and show that the common progression mutations modify Bim levels and sensitivity to TGF-β during intestinal adenoma development.

High PROX1 expression in gastric cancer predicts better survival

Background PROX1 is a transcription factor involved in the development of various organs. It has also an important function in colorectal cancer progression. The aim of this study was to investigate the prognostic role of PROX1 expression in gastric cancer. Methods We evaluated PROX1 expression in gastric cancer by immunohistochemistry of tumor-tissue microarrays including tumor specimens from 283 patients who underwent surgery at Helsinki University Hospital. We investigated the association of PROX1 expression with clinicopathologic variables and patient survival. Results Cytoplasmic PROX1 reactivity was high in 56 (20.5%) and low in 217 (79.5%) cases. Low PROX1 immunostaining associated with diffuse cancer type (p = 0.002). In subgroup analysis, PROX1 was a significant marker of better prognosis in patients aged under 66 (p = 0.007), in those with intestinal cancer (p = 0.025), among men (p = 0.019), and in tumors of less than 5 cm diameter (p = 0.030). Patients with high PROX1 expression had a cancer-specific 5-year survival of 65.6% (95% CI 52.7–78.5), compared to 37.1% (95% CI 30.2–44.0) for those with low expression (p = 0.004, log-rank test). This result remained significant in multivariable analysis (HR = 0.56; 95% CI 0.35–0.90; p = 0.017). Conclusion In gastric cancer, high cytoplasmic PROX1 expression is an independent marker of better prognosis.

Transcription Factor PROX1 Suppresses Notch Pathway Activation via the Nucleosome Remodeling and Deacetylase Complex in Colorectal Cancer Stem–like Cells

The homeobox transcription factor PROX1 is induced by high Wnt/β-catenin activity in intestinal adenomas and colorectal cancer, where it promotes tumor progression. Here we report that in LGR5+ colorectal cancer cells, PROX1 suppresses the Notch pathway, which is essential for cell fate in intestinal stem cells. Pharmacologic inhibition of Notch in ex vivo 3D organoid cultures from transgenic mouse intestinal adenoma models increased Prox1 expression and the number of PROX1-positive cells. Notch inhibition led to increased proliferation of the PROX1-positive colorectal cancer cells, but did not affect their ability to give rise to PROX1-negative secretory cells. Conversely, PROX1 deletion increased Notch target gene expression and NOTCH1 promoter activity, indicating reciprocal regulation between PROX1 and the Notch pathway in colorectal cancer. PROX1 interacted with the nucleosome remodeling and deacetylase (NuRD) complex to suppress the Notch pathway. Thus, our data suggests that PROX1 and Notch suppress each other and that PROX1-mediated suppression of Notch mediates its stem cell function in colorectal cancer.Significance: These findings address the role of the PROX1 homeobox factor as a downstream effector of Wnt/β-catenin singling in colorectal cancer stem cells and show that PROX1 inhibits the Notch pathway and helps to enforce the stem cell phenotype and inhibit differentiation. Cancer Res; 78(20); 5820-32. ©2018 AACR.

Abstract 2320: Prox1 marks a stem cell population that promotes tumor progression in intestinal adenomas

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of this cell population. We have shown that a subpopulation of cells expressing the Prox1 transcription factor have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth in multiple CRC model systems. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut. To further study the adenoma stem cells in relation to the Prox1-positive cell population, we analyzed Apc-mutant intestinal organoids derived from Prox1-CreER; tdTomatoflox/Stop/flox; Apcmin/+ mice. In this lineage tracing model, the induction of Cre activity results in the expression of the red fluorescent protein tdTomato only in the Prox1-positive adenoma cells. FACS sorted tdTomato-positive cell population displayed a highly elevated capability to form new organoids compared to the tdTomato-negative cells, consistent with stem cells enrichment in the Prox1-positive cell population. Gene expression analysis provided new clues about the identity of the Prox1 positive cells. Citation Format: Zoltan Wiener, Jenny Hogstrom, Ville Hyvonen, Pauliina Kallio, Sarika Heino, Kari Alitalo. Prox1 marks a stem cell population that promotes tumor progression in intestinal adenomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2320. doi:10.1158/1538-7445.AM2015-2320

Abstract 1908: The Wnt-target Prox1 promotes colorectal cancer stem cell survival to fuel tumor growth

Colorectal cancer (CRC) is one of the leading causes of cancer mortality in the Western countries. In most of the CRC patients, an initial mutation occurs in the APC or CTNNB1 genes, leading to the ligand-independent activation of the canonical Wnt pathway. Besides having a central role in the development of CRC, the Wnt pathway plays a critical role also in the maintenance of the normal intestine. Attempts at therapeutic inhibition of this pathway could thus lead to serious side effects in CRC patients. We have previously shown that the Prox1 transcription factor is induced in the intestinal epithelium by mutations activating the Wnt pathway and it critically contributes to CRC progression via an unknown mechanism. Here we provide evidence that Prox1 expression is induced in the Lgr5+ adenoma stem cells early after Apc deletion. Our in vivo models and ex vivo organoid experiments suggest that Prox1 silencing or deletion restricts the expansion of the Lgr5+ adenoma stem cell population both in humans and in mice. Interestingly, silencing the phospholipid binding protein Annexin A1 (Anxa1), a gene suppressed by Prox1, is responsible for several of the effects of Prox1 on adenoma cells, such as the re-organization of the actin cytoskeleton, enhanced stem cell activity and tumor growth. Furthermore, Prox1 deletion abnormally increases the mTORC1 pathway activity, which results in decreased survival of the adenoma stem cells. Since Prox1 is expressed at low level only in rare neuroendocrine cells and in some Paneth cells in the wild type intestinal epithelium, furthermore, its genetic deletion in the adult gut does not lead to obvious phenotypes, Prox1 may serve as an attractive therapeutic target for restricting the progression of early intestinal adenomas. Citation Format: Zoltan Wiener, Ville Hyvonen, Jenny Hogstrom, Tanja Holopainen, Arja Band, Pauliina Kallio, Olli Dufva, Caj Haglund, Olli Kruuna, Guillermo Oliver, Yinon Ben-Neriah, Kari Alitalo. The Wnt-target Prox1 promotes colorectal cancer stem cell survival to fuel tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1908. doi:10.1158/1538-7445.AM2014-1908