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Membrane Potential and Stem Cell Potency in Normal and Malignant Tissues

Periodic Reporting for period 1 - MPSC (Membrane Potential and Stem Cell Potency in Normal and Malignant Tissues)

Reporting period: 2016-04-01 to 2018-03-31

The studies proposed in the current application seek to answer a critical question hindering progress in the early detection and treatment of cancer: How is normal differentiation and lineage commitment disrupted in the earliest stages of cancer development? Better understanding of the processes that govern differentiation and lineage commitment hold promise to advance the fields of regenerative medicine and oncology. As a first step to answer this question we have generated a multi-organ mouse cancer and lineage tracing model by recombining conditional oncogene, tumour suppressor gene and lineage tracing alleles in cells expressing Cre-recombinase from the Promin1 locus: Promin1 marks stem and progenitor cells in several organs. Focusing on the gastric mucosa we showed that this tissue is regenerated by normal Promin1 stem cells and forms gastric cancer when Kras and Tp53 mutations are introduced into these cells. Remarkably, comparison of the transcriptomes of normal and malignant Promin1+ gastric stem cells revealed a selective silencing of ion channels and solute carriers in the malignant stem population. One of these genes, Nalcn, encodes the ion channel responsible for the resting membrane potential of cells. Here, I will test the hypothesis that ion-channel regulated membrane potential dictates normal and gastric cancer stem cell differentiation capacity. Manipulation of this process might prove a useful therapeutic approach.

Project objectives:
1. Determine the role of Nalcn in stem cell biology/normal development
2. Determine the role of Nalcn in tumourigenesis

Conclusions of the action:
Our data demonstrate that Nalcn has a tumour suppressive role for many cancers including gastric, epididymal, bladder and lung. Moreover, a cell autonomous reduction in Nalcn expression significantly increased non-cell autonomous immune infiltration and inflammation in numerous tissue types. Collectively these data suggest new roles for the regulation of membrane potential in controlling cell-fate choices and inflammation. Manipulation of this process might prove a useful therapeutic approach.
Overview of the work:
The initial phases of the project focused on two points 1) spatial/temporal profiling of Nalcn expression body-wide and 2) characterization of the conditional Nalcn allele mouse model. In order to determine the spatial/temporal profiling of Nalcn expression, I evaluated a variety of molecular (qRT-PCR) and histological approaches (in situ hybridization and immunohistochemistry) to accurately identify Nalcn expressing cells in E9.5-E18 and postnatal D1 and D60 tissue samples. These experiments lead to the development of new in situ hybridization probes, identification of numerous inaccurate antibodies and one reliably validated antibody. Using these tools, we determined spatial/temporal profiling of Nalcn. We observed similar results to what was described in the literature, but we were able to further characterize the localization of Nalcn to a variety of stem cells in multiple tissues throughout body including stomach, small intestine, colon, brain and skin.
The next steps of our experiments involved devising a series of strategic genetic mouse model crosses to determine the role of Nalcn in normal development and tumourigenesis. We used the Prominin-1 driven Cre recombinase mouse model to delete Nalcn in stem cells body-wide. We combined this with a conditional reporter allele, ZSGreen, to identify targeted cells histologically. For understanding the role of Nalcn in normal development, we generated experimental (Prom1C-L/+;R26ZSGreen;Nalcnf/+ , Prom1C-L/+;R26ZSGreen;Nalcnf/f) and control (Prom1C-L/+;R26ZSGreen) mouse cohorts. To induce Nalcn loss, animals received two intraperitoneal injections of tamoxifen at postnatal time points P1/2 (development) or P60/61 (homeostasis) then aged and monitored for phenotypic alterations. Additionally, we crossed known oncogenes (KRASG12D) and tumour suppressor genes (Trp53f/f) to the conditional allele to asses the impact on tumour formation. Our preliminary data indicate Prom1C-L;KRASG12D;Trp53f/f mice develop gastric cancers from Prom1+ malignant stem cells. Tamoxifen injected animals (neonatal or adult) are being aged and sacrificed, tissues harvested at designated time points or at first signs of illness for histological analysis and transcriptomic profiling.
Our preliminary histological analysis of tissues from Nalcn modified animals at several time points post tamoxifen induction (30d, 90d, 180d, 270d, 365d) in our developmental models demonstrated that Nalcn loss alone has an important role in normal cellular development and a tumour suppressive role for many tissue types. We observed maldifferentiation in the GI tract, fibrosis in several tissues body-wide and tumour formation. Moreover, reduction in Nalcn expression cell autonomously significantly increased non-cell autonomous immune infiltration and inflammation in several tissues including the GI tract. In our tumour background models, Nalcn loss has enhanced tumour penetrance, metastasis and increases inflammation. We are currently carrying out additional in depth histological profiling of these tissues to better resolve the phenotypes associated with Nalcn loss.
In order to better understand the mechanism of Nalcn in the GI tract, we carried out transcriptomic profiling of GI tract tissues from 9-month old animals with no overt histological phenotype. Analysis of the stomach data identified a pro-inflammatory signature and a panel of genes significantly differentially expressed in human gastric cancer. Further in depth analysis is required and we are expanding the transcriptomic profiling to early stages and additional tissues (e.g. kidney, epididymis).

Exploitation and dissemination:
This work has presented and discussed at variety of venues including the CRUK Cambridge Institute seminar series, the Cambridge Cancer Centre Symposium, the International Society for Stem Cell Research, Cell Symposia Cancer, Inflammation and Immunity and the Cell Symposia.
Previous data strongly implicate the proper maintenance of membrane potential in normal and malignant cell differentiation but the precise roles and genes involved in this process and metastatic progression is unknown. In this proposal we altered membrane potential in Prom1+ cells in normal and tumorigenic backgrounds in vivo and histologically. To our knowledge, these are the first studies using genetically engineered mouse models to target specific cellular compartments of the GI tract to interrogate the role of membrane potential in stem cell function and cell fate. We have uncovered a novel mechanism by which cell autonomous alterations in membrane potential lead to altered cell fate (tumourigenesis) and a non-cell autonomous inflammation. It is possible these mechanisms are tightly linked due to perturbations in the local microenvironment. Further experiments assessing these early stage alterations prior to tumour formation will shed light on new potential early diagnostic markers and possible therapeutic intervention.
The impact of Nalcn loss on normal and malignant development.