Targeting the Wnt Pathway and Cancer Stem Cells with Anti-progastrin Humanized Antibodies as a Potential Treatment for K-RAS-Mutated Colorectal Cancer.

Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct ß-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses.Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling.Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors.Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS-mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267-80. ©2017 AACR.

The p53 isoform Δ133p53ß promotes cancer stem cell potential.

Cancer stem cells (CSC) are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53ß, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53ß stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53ß expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53ß in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53ß supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53ß isoform.

An unusual DNA binding compound, S23906, induces mitotic catastrophe in cultured human cells.

The biochemical pathways that lead cells to mitotic catastrophe are not well understood. To identify these pathways, we have taken an approach of treating cells with a novel genotoxic compound and characterizing whether cells enter mitotic catastrophe or not. S23906 is a novel acronycine derivative that forms adducts with the N2 residue of guanine in the minor groove of the DNA helix and destabilizes base pairing to cause helix opening. We observed, in HeLa and HT-29 cells, that S23906 induced gamma-H2AX and activated checkpoint kinase 1, as did bleomycin, camptothecin, and cisplatin, when tested under equi-toxic conditions. S23906 also induced cyclin E1 protein, although this activity was not required for cytotoxicity because knock down of cyclin E1 by RNA interference did not affect the number of dead cells after treatment. Cyclin B1 levels first decreased and then increased after treatment with S23906. Cyclin B1 was associated with Cdk1 kinase activity, which correlated with an increase in the number of mitotic cells. By 32 h after treatment, at least 20% of the cells entered mitotic catastrophe as determined by microscopy. Suppression of the DNA checkpoint response by co-treatment with caffeine increased the number of cells in mitosis. These results suggest that mitotic catastrophe is one of the cellular responses to S23906 and that mitotic catastrophe may be a common cellular response to many different types of DNA damage.

CD44 is of functional importance for colorectal cancer stem cells.

PURPOSE: Both CD44 and CD133 were reported as putative markers for isolating colorectal cancer stem cells (CSC). It remains to be resolved if both of these markers are of functional importance for colorectal CSC. EXPERIMENTAL DESIGN: The expression of CD44 and CD133 in normal colonic tissues and primary colorectal cancer was assessed by immunohistochemistry in a series of 60 patients on tissue microarray sections. Both in vitro clonogenic and in vivo tumorigenic assay were applied to measure CSC activities from the cells isolated from patients. Lentiviral RNA interference was used to stably knock down CD44 or CD133 in colorectal cancer cells from patients. RESULTS: We found that CD44(+) cells displayed clustered growth and they did not colocalize with CD133(+) cells within colorectal cancer. As few as 100 CD44(+) cells from a patients' tumor initiated a xenograft tumor in vivo. A single CD44(+) cell from a tumor could form a sphere in vitro which has characteristic stem cell properties and was able to generate a xenograft tumor resembling the properties of the primary tumor. Knockdown of CD44, but not CD133, strongly prevented clonal formation and inhibited tumorigenicity in xenograft model. CONCLUSIONS: These results indicate that CD44 is a robust marker and is of functional importance for colorectal CSC for cancer initiation.

Lipid raft localization and palmitoylation: identification of two requirements for cell death induction by the tumor suppressors UNC5H.

UNC5H receptors (UNC5H1, UNC5H2, UNC5H3) are putative tumor suppressors whose expression is lost in numerous cancers. These receptors have been shown to belong to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand netrin-1 is absent, thus conferring a state of cellular dependence towards ligand presence. Along this line, these receptors may limit tumor progression because they induce the death of tumor cells that grow in settings of ligand unavailability. We show here that UNC5H receptors are localized to cholesterol-and sphingolipid-enriched membrane domains called lipid rafts. We then demonstrate that the lipid raft localization of UNC5H2 is required for the pro-apoptotic activity of unbound UNC5H2. We also propose that this lipid raft localization is probably mediated via the recruitment of adaptor protein(s) within the death domain of UNC5H2 but is not dependent on the post-translational modification by palmitoylation of UNC5H2 even though this palmitoylation is required for UNC5H2 pro-apoptotic activity. Moreover we show that the interaction of UNC5H2 with the downstream pro-apoptotic serine threonine kinase DAPk is dependent on both UNC5H2 lipid raft localization and palmitoylation. Thus, we propose that the UNC5H dependence receptors require lipid raft localization and palmitoylation to trigger apoptosis.

Silencing of prion protein sensitizes breast adriamycin-resistant carcinoma cells to TRAIL-mediated cell death.

We investigated the relationship between the resistance to the proapoptotic action of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and cellular prion protein (PrPc) function, using the TRAIL-sensitive MCF-7 human breast adenocarcinoma cell line and two TRAIL-resistant sublines: 2101 and MCF-7/ADR. All of the cell lines tested expressed TRAIL-R1 and TRAIL-R2. TRAIL decoy receptors were not detected, suggesting that the resistance of 2101 and MCF-7/ADR cells, strongly expressing PrPc, to TRAIL-mediated cell death was independent from the expression of TRAIL receptors and death-inducing signaling complex formation. Down-regulation of PrPc by small interfering RNA increased the sensitivity of Adriamycin- and TRAIL-resistant cells to TRAIL, but not to epirubicin/Adriamycin. TRAIL-mediated apoptosis in PrPc knocked-down cells was associated with caspase processing, Bid cleavage, and Mcl-1 degradation. In addition, an increased sensitivity of apoptosis-resistant cells to TRAIL after PrPc silencing was not associated with the increased recruitment of receptors and intracellular signaling molecule to the death-inducing signaling complex. Bcl-2 expression was substantially decreased after PrPc knock-down but the levels of Bcl-X(L) and Mcl-1 were not affected. The down-regulation of Bcl-2 was concomitant with Bax delocalization. Our findings support the notion that silencing of PrPc facilitates the activation of proapoptotic Bax by down-regulation of Bcl-2 expression, thereby abolishing the resistance of breast cancer cells to TRAIL-induced apoptosis.

The dependence receptor DCC requires lipid raft localization for cell death signaling.

DCC (deleted in colorectal cancer) is a putative tumor suppressor gene whose expression is lost in numerous cancers. DCC also encodes the main receptor for the neuronal navigation cue netrin-1. It has been shown that DCC belongs to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand is absent, thus conferring a state of cellular dependence on ligand availability. We recently proposed that DCC is a tumor suppressor because it induces the death of tumor cells that grow in settings of ligand unavailability. Moreover, it seems that the DCC/netrin-1 pair may also regulate neuron survival during nervous system development. However, the mechanisms by which DCC triggers cell death are still unknown. We show here that the localization of DCC to lipid rafts is a prerequisite for its proapoptotic activity, both in immortalized cells and in primary neurons. The presence of DCC in lipid rafts probably allows the formation of an adequate submembrane complex, because the interaction of caspase-9 with DCC is inhibited by the disorganization of lipid rafts. Thus, dependence receptors may require lipid raft localization for cell death signaling.

Fas ligand is localized to membrane rafts, where it displays increased cell death-inducing activity.

Fas ligand (FasL), a member of the TNF protein family, potently induces cell death by activating its matching receptor Fas. Fas-mediated killing plays a critical role in naturally and pathologically occurring cell death, including development and homeostasis of the immune system. In addition to its receptor-interacting and cell death-inducing extracellular domain, FasL has a well-conserved intracellular portion with a proline-rich SH3 domain-binding site probably involved in non-apoptotic functions. We report here that, as with the Fas receptor, a fraction of FasL is constitutively localized in rafts. These dynamic membrane microdomains, enriched in sphingolipids and cholesterol, are important for cell signaling and trafficking processes. We show that FasL is partially localized in rafts and that increased amounts of FasL are found in rafts after efficient FasL/Fas receptor interactions. Raft disorganization after cholesterol oxidase treatment and deletions within the intracellular FasL domain diminish raft partitioning and, most important, lead to decreased FasL killing. We conclude that FasL is recruited into lipid rafts for maximum Fas receptor contact and cell death-inducing potency. These findings raise the possibility that certain pathologic conditions may be treated by altering the cell death-inducing capability of FasL with drugs affecting its raft localization.

DCC association with lipid rafts is required for netrin-1-mediated axon guidance.

During development, axons migrate long distances in responses to attractive or repulsive signals that are detected by their growth cones. One of these signals is mediated by netrin-1, a diffusible laminin-related molecule that both attracts and repels growth cones via interaction with its receptor DCC (deleted in colorectal cancer). Here we show that DCC in both commissural neurons and immortalized cells, is partially associated with cholesterol- and sphingolipid-enriched membrane domains named lipid rafts. This localization of DCC in lipid rafts is mediated by the palmitoylation within its transmembrane region. Moreover, this raft localization of DCC is required for netrin-1-induced DCC-dependent ERK activation, and netrin-1-mediated axon outgrowth requires lipid raft integrity. Thus, the presence of axon guidance-related receptors in lipid rafts appears to be a crucial pre-requisite for growth cone response to chemo-attractive or repulsive cues.