CFTR and Gastrointestinal Cancers: An Update.

Cystic Fibrosis (CF) is a disease caused by mutations in the CFTR gene that severely affects the lungs as well as extra-pulmonary tissues, including the gastrointestinal (GI) tract. CFTR dysfunction resulting from either mutations or the downregulation of its expression has been shown to promote carcinogenesis. An example is the enhanced risk for several types of cancer in patients with CF, especially cancers of the GI tract. CFTR also acts as a tumor suppressor in diverse sporadic epithelial cancers in many tissues, primarily due to the silencing of CFTR expression via multiple mechanisms, but especially due to epigenetic regulation. This review provides an update on the latest research linking CFTR-deficiency to GI cancers, in both CF patients and in sporadic GI cancers, with a particular focus on cancer of the intestinal tract. It will discuss changes in the tissue landscape linked to CFTR-deficiency that may promote cancer development such as breakdowns in physical barriers, microbial dysbiosis and inflammation. It will also discuss molecular pathways and mechanisms that act upstream to modulate CFTR expression, such as by epigenetic silencing, as well as molecular pathways that act downstream of CFTR-deficiency, such as the dysregulation of the Wnt/ß-catenin and NF-κB signaling pathways. Finally, it will discuss the emerging CFTR modulator drugs that have shown promising results in improving CFTR function in CF patients. The potential impact of these modulator drugs on the treatment and prevention of GI cancers can provide a new example of personalized cancer medicine.

Cancer spectrum in TP53-deficient golden Syrian hamsters: A new model for Li-Fraumeni syndrome.

BACKGROUND: The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. Humans who inherit mutant TP53 alleles develop a wide range of early onset cancers, a disorder called Li-Fraumeni Syndrome (LFS). Trp53-deficient mice recapitulate most but not all of the cancer phenotypes observed in TP53-deficient human cancers, indicating that new animal models may complement current mouse models and better inform on human disease development. MATERIALS AND METHODS: The recent application of CRISPR/Cas9 genetic engineering technology has permitted the emergence of golden Syrian hamsters as genetic models for wide range of diseases, including cancer. Here, the first cancer phenotype of TP53 knockout golden Syrian hamsters is described. RESULTS: Hamsters that are homozygous for TP53 mutations become moribund on average ~ 139 days of age, while hamsters that are heterozygous become moribund at ~ 286 days. TP53 homozygous knockout hamsters develop a wide range of cancers, often synchronous and metastatic to multiple tissues, including lymphomas, several sarcomas, especially hemangiosarcomas, myeloid leukemias and several carcinomas. TP53 heterozygous mutants develop a more restricted tumor spectrum, primarily lymphomas. CONCLUSIONS: Overall, hamsters may provide insights into how TP53 deficiency leads to cancer in humans and can become a new model to test novel therapies.

Role of ion channels in gastrointestinal cancer.

In their seminal papers Hanahan and Weinberg described oncogenic processes a normal cell undergoes to be transformed into a cancer cell. The functions of ion channels in the gastrointestinal (GI) tract influence a variety of cellular processes, many of which overlap with these hallmarks of cancer. In this review we focus on the roles of the calcium (Ca2+), sodium (Na+), potassium (K+), chloride (Cl-) and zinc (Zn2+) transporters in GI cancer, with a special emphasis on the roles of the KCNQ1 K+ channel and CFTR Cl- channel in colorectal cancer (CRC). Ca2+ is a ubiquitous second messenger, serving as a signaling molecule for a variety of cellular processes such as control of the cell cycle, apoptosis, and migration. Various members of the TRP superfamily, including TRPM8, TRPM7, TRPM6 and TRPM2, have been implicated in GI cancers, especially through overexpression in pancreatic adenocarcinomas and down-regulation in colon cancer. Voltage-gated sodium channels (VGSCs) are classically associated with the initiation and conduction of action potentials in electrically excitable cells such as neurons and muscle cells. The VGSC NaV1.5 is abundantly expressed in human colorectal CRC cell lines as well as being highly expressed in primary CRC samples. Studies have demonstrated that conductance through NaV1.5 contributes significantly to CRC cell invasiveness and cancer progression. Zn2+ transporters of the ZIP/SLC39A and ZnT/SLC30A families are dysregulated in all major GI organ cancers, in particular, ZIP4 up-regulation in pancreatic cancer (PC). More than 70 K+ channel genes, clustered in four families, are found expressed in the GI tract, where they regulate a range of cellular processes, including gastrin secretion in the stomach and anion secretion and fluid balance in the intestinal tract. Several distinct types of K+ channels are found dysregulated in the GI tract. Notable are hERG1 upregulation in PC, gastric cancer (GC) and CRC, leading to enhanced cancer angiogenesis and invasion, and KCNQ1 down-regulation in CRC, where KCNQ1 expression is associated with enhanced disease-free survival in stage II, III, and IV disease. Cl- channels are critical for a range of cellular and tissue processes in the GI tract, especially fluid balance in the colon. Most notable is CFTR, whose deficiency leads to mucus blockage, microbial dysbiosis and inflammation in the intestinal tract. CFTR is a tumor suppressor in several GI cancers. Cystic fibrosis patients are at a significant risk for CRC and low levels of CFTR expression are associated with poor overall disease-free survival in sporadic CRC. Two other classes of chloride channels that are dysregulated in GI cancers are the chloride intracellular channels (CLIC1, 3 & 4) and the chloride channel accessory proteins (CLCA1,2,4). CLIC1 & 4 are upregulated in PC, GC, gallbladder cancer, and CRC, while the CLCA proteins have been reported to be down-regulated in CRC. In summary, it is clear, from the diverse influences of ion channels, that their aberrant expression and/or activity can contribute to malignant transformation and tumor progression. Further, because ion channels are often localized to the plasma membrane and subject to multiple layers of regulation, they represent promising clinical targets for therapeutic intervention including the repurposing of current drugs.

A novel cancer syndrome caused by KCNQ1-deficiency in the golden Syrian hamster.

BACKGROUND: The golden Syrian hamster is an emerging model organism. To optimize its use, our group has made the first genetically engineered hamsters. One of the first genes that we investigated is KCNQ1 which encodes for the KCNQ1 potassium channel and also has been implicated as a tumor suppressor gene. MATERIALS AND METHODS: We generated KCNQ1 knockout (KO) hamsters by CRISPR/Cas9-mediated gene targeting and investigated the effects of KCNQ1-deficiency on tumorigenesis. RESULTS: By 70 days of age seven of the eight homozygous KCNQ1 KOs used in this study began showing signs of distress, and on necropsy six of the seven ill hamsters had visible cancers, including T-cell lymphomas, plasma cell tumors, hemangiosarcomas, and suspect myeloid leukemias. CONCLUSIONS: None of the hamsters in our colony that were wild-type or heterozygous for KCNQ1 mutations developed cancers indicating that the cancer phenotype is linked to KCNQ1-deficiency. This study is also the first evidence linking KCNQ1-deficiency to blood cancers.

TRAP1 regulates autophagy in lung cancer cells.

BACKGROUND: Expression of TRAP1, a member of the HSP90 chaperone family, has been implicated in tumour protective effects, based on its differential mitochondrial localization and function. DESIGN: This work was designed to provide new insights into the pathways involved in TRAP1-provided cytoprotection on NSCLC. For this, TRAP1-depleted A549 human NSCLC cells and MRC-5 normal lung fibroblasts were produced using a siRNA approach and main cellular quality control mechanisms were investigated. RESULTS: TRAP1-depleted A549 cells displayed decreased cell viability likely due to impaired mitochondrial function including decreased ATP/AMP ratio, oxygen consumption and membrane potential, as well as increased apoptotic indicators. Furthermore, the negative impact of TRAP1 depletion on mitochondrial function was not observed in normal MRC-5 lung cells, which might be due to the differential intracellular localization of the chaperone in tumour versus normal cells. Additionally, A549 TRAP1-depleted cells showed increased autophagic flux. Functionally, autophagy inhibition resulted in decreased cell viability in both TRAP1-expressing and TRAP1-depleted tumour cells with minor effects on MRC-5 cells. Conversely, autophagy stimulation decreased cell viability of both A549 and MRC-5 TRAP1-expressing cells while in A549 TRAP1-depleted cells, increased autophagy augmented viability. CONCLUSIONS: Our results show that even though TRAP1 depletion affects both normal MRC-5 and tumour A549 cell proliferation, inhibition of autophagy per se led to a decrease in tumour cell mass, while having a reduced effect on the normal cell line. The strategy of targeting TRAP1 in NSCLC shows future potential therapeutic applications.

Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide. Accurately identifying stage II CRC patients at risk for recurrence is an unmet clinical need. KCNQ1 was previously identified as a tumour suppressor gene and loss of expression was associated with poor survival in patients with CRC liver metastases. In this study the prognostic value of KCNQ1 in stage II and stage III colon cancer patients was examined. METHODS: KCNQ1 mRNA expression was assessed in 90 stage II colon cancer patients (AMC-AJCCII-90) using microarray gene expression data. Subsequently, KCNQ1 protein expression was evaluated in an independent cohort of 386 stage II and stage III colon cancer patients by immunohistochemistry of tissue microarrays. RESULTS: Low KCNQ1 mRNA expression in stage II microsatellite stable (MSS) colon cancers was associated with poor disease-free survival (DFS) (P=0.025). Loss of KCNQ1 protein expression from epithelial cells was strongly associated with poor DFS in stage II MSS (P<0.0001), stage III MSS (P=0.0001) and stage III microsatellite instable colon cancers (P=0.041). KCNQ1 seemed an independent prognostic value in addition to other high-risk parameters like angio-invasion, nodal stage and microsatellite instability-status. CONCLUSIONS: We conclude that KCNQ1 is a promising biomarker for prediction of disease recurrence and may aid stratification of patients with stage II MSS colon cancer for adjuvant chemotherapy.

Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.

The intestinal stem cell niche provides cues that actively maintain gut homeostasis. Dysregulation of these cues may compromise intestinal regeneration upon tissue insult and/or promote tumor growth. Here, we identify secreted phospholipases A2 (sPLA2s) as stem cell niche factors with context-dependent functions in the digestive tract. We show that group IIA sPLA2, a known genetic modifier of mouse intestinal tumorigenesis, is expressed by Paneth cells in the small intestine, while group X sPLA2 is expressed by Paneth/goblet-like cells in the colon. During homeostasis, group IIA/X sPLA2s inhibit Wnt signaling through intracellular activation of Yap1. However, upon inflammation they are secreted into the intestinal lumen, where they promote prostaglandin synthesis and Wnt signaling. Genetic ablation of both sPLA2s improves recovery from inflammation but increases colon cancer susceptibility due to release of their homeostatic Wnt-inhibitory role. This "trade-off" effect suggests sPLA2s have important functions as genetic modifiers of inflammation and colon cancer.

Mitochondrial remodeling in cancer metabolism and survival: potential for new therapies.

Mitochondria are semi-autonomous organelles that play essential roles in cellular metabolism and programmed cell death pathways. Genomic, functional and structural mitochondrial alterations have been associated with cancer. Some of those alterations may provide a selective advantage to cells, allowing them to survive and grow under stresses created by oncogenesis. Due to the specific alterations that occur in cancer cell mitochondria, these organelles may provide promising targets for cancer therapy. The development of drugs that specifically target metabolic and mitochondrial alterations in tumor cells has become a matter of interest in recent years, with several molecules undergoing clinical trials. This review focuses on the most relevant mitochondrial alterations found in tumor cells, their contribution to cancer progression and survival, and potential usefulness for stratification and therapy.

Runx1 is a tumor suppressor gene in the mouse gastrointestinal tract.

The Runx1 transcription factor plays an important role in tissue homeostasis through its effects on stem/progenitor cell populations and differentiation. The effect of Runx1 on epithelial differentiation of the secretory cell lineage of the colon was recently demonstrated. This study aimed to examine the role of Runx1 in tumor development in epithelial cells of the gastrointestinal tract. Conditional knockout mice that lacked Runx1 expression in epithelial cells of the GI tract were generated. These mice were crossed onto the Apc(Min) background, killed and their intestinal tumor phenotypes were compared with Apc(Min) Runx1 wild-type control mice. Apc-wild-type Runx1-mutant mice were also examined for tumor development. Colons from Runx1 knockout and wild-type mice were used for genome-wide mRNA expression analyses followed by gene-specific quantitative RT-PCR of whole colon and colon epithelium to identify Runx1 target genes. Runx1 deficiency in intestinal epithelial cells significantly enhanced tumorigenesis in Apc(Min) mice. Notably, epithelial Runx1 deficiency in Apc-wild-type mice was sufficient to cause tumor development. Absence of Runx1 was associated with global changes in the expression of genes involved in inflammation and intestinal metabolism, and with gene sets indicative of a metastatic phenotype and poor prognosis. Gene-specific analysis of Runx1-deficient colon epithelium revealed increased expression of genes linked to an expansion of the stem/progenitor cell population. These results identify Runx1 as a novel tumor suppressor gene for gastrointestinal tumors and support a role for Runx1 in maintaining the balance between the intestinal stem/progenitor cell population and epithelial differentiation of the GI tract.

A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis.

It is proposed that a progressive series of mutations and epigenetic events leads to human colorectal cancer (CRC) and metastasis. Furthermore, data from resequencing of the coding regions of human CRC suggests that a relatively large number of mutations occur in individual human CRC, most at low frequency. The functional role of these low-frequency mutations in CRC, and specifically how they may cooperate with high-frequency mutations, is not well understood. One of the most common rate-limiting mutations in human CRC occurs in the adenomatous polyposis coli (APC) gene. To identify mutations that cooperate with mutant APC, we performed a forward genetic screen in mice carrying a mutant allele of Apc (Apc(Min)) using Sleeping Beauty (SB) transposon-mediated mutagenesis. Apc(Min) SB-mutagenized mice developed three times as many polyps as mice with the Apc(Min) allele alone. Analysis of transposon common insertion sites (CIS) identified the Apc locus as a major target of SB-induced mutagenesis, suggesting that SB insertions provide an efficient route to biallelic Apc inactivation. We also identified an additional 32 CIS genes/loci that may represent modifiers of the Apc(Min) phenotype. Five CIS genes tested for their role in proliferation caused a significant change in cell viability when message levels were reduced in human CRC cells. These findings demonstrate the utility of using transposon mutagenesis to identify low-frequency and cooperating cancer genes; this approach will aid in the development of combinatorial therapies targeting this deadly disease.

Synthesis of α-carboranyl-α-acyloxy-amides as potential BNCT agents.

Novel α-carboranyl-α-acyloxy-amides were prepared as potential BNCT agents utilizing three component Passerini reaction. Preliminary cytotoxicity of the representative compounds on two brain tumor cell lines (U-87 and A-172) showed no effect on cell viability; an essential requirement for utility as potential BNCT agents.

GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network.

Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.