A clinically relevant heterozygous ATR mutation sensitizes colorectal cancer cells to replication stress.

Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

Tumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis.

Ki-67 is a nuclear protein that is expressed in all proliferating vertebrate cells. Here, we demonstrate that, although Ki-67 is not required for cell proliferation, its genetic ablation inhibits each step of tumor initiation, growth, and metastasis. Mice lacking Ki-67 are resistant to chemical or genetic induction of intestinal tumorigenesis. In established cancer cells, Ki-67 knockout causes global transcriptome remodeling that alters the epithelial-mesenchymal balance and suppresses stem cell characteristics. When grafted into mice, tumor growth is slowed, and metastasis is abrogated, despite normal cell proliferation rates. Yet, Ki-67 loss also down-regulates major histocompatibility complex class I antigen presentation and, in the 4T1 syngeneic model of mammary carcinoma, leads to an immune-suppressive environment that prevents the early phase of tumor regression. Finally, genes involved in xenobiotic metabolism are down-regulated, and cells are sensitized to various drug classes. Our results suggest that Ki-67 enables transcriptional programs required for cellular adaptation to the environment. This facilitates multiple steps of carcinogenesis and drug resistance, yet may render cancer cells more susceptible to antitumor immune responses.

Ascorbic acid is a dose-dependent inhibitor of adipocyte differentiation, probably by reducing cAMP pool.

Ascorbic acid (AA) is the active component of vitamin C and antioxidant activity was long considered to be the primary molecular mechanism underlying the physiological actions of AA. We recently demonstrated that AA is a competitive inhibitor of adenylate cyclase, acting as a global regulator of intracellular cyclic adenosine monophosphate (cAMP) levels. Our study, therefore, aimed to determine new targets of AA that would account for its potential effect on signal transduction, particularly during cell differentiation. We demonstrated that AA is an inhibitor of pre-adipocyte cell line differentiation, with a dose-dependent effect. Additionally, we describe the impact of AA on the expression of genes involved in adipogenesis and/or the adipocyte phenotype. Moreover, our data suggest that treatment with AA partially reverses lipid accumulation in mature adipocytes. These properties likely reflect the function of AA as a global regulator of the cAMP pool, since an analog of AA without any antioxidant properties elicited the same effect. Additionally, we demonstrated that AA inhibits adipogenesis in OP9 mesenchymal cell line and drives the differentiation of this line toward osteogenesis. Finally, our data suggest that the intracellular transporter SVCT2 is involved in these processes and may act as a receptor for AA.

CamKII inhibitors reduce mitotic instability, connexon anomalies and progression of the in vivo behavioral phenotype in transgenic animals expressing a mutated Gjb1 gene.

Mutation in the Gjb1 gene, coding for a connexin (Cx32), is associated with an inherited peripheral neuropathic disorder (X-linked Charcot-Marie-Tooth, CMTX). Our previous work reported that transgenic animals expressing a human Gjb1 transgene present polyploidy and abnormal over-duplication of the centrosome, suggesting a role for Gjb1 in mitotic stability. In this article, we propose mechanisms by which mutations in Gjb1 induce mitotic instability and discuss its potential relation with the CMTX phenotype. We showed that transgenic cells exhibit CamKII over-stimulation, a phenomenon that has been linked to mitotic instability (polyploidy, nuclear volume and centrosome over-duplication), that is reversed by CamKII inhibitors. We also demonstrate that connexon activity is partially restored in transgenic cells with CamKII inhibitors. Our model supports the role for Pim1, a kinase that has been associated with genomic instability in cancers, in genomic instability in Cx32 mutations. Regarding in vivo phenotype, we showed that degradation on the rotarod test in our transgenic mice is significantly lowered by treatment with a CamKII inhibitor (KN93). This effect was seen in two lines with different point mutations in GJB1, and stopping the treatment led to degradation of the phenotype.

Ascorbic acid derivatives as a new class of antiproliferative molecules.

Ascorbic acid (AA) has long been described as an antiproliferative agent. However, the molecule has to be used at a very high concentrations, which necessitates i.v. injection, and the tight regulation of in-blood and in-cell AA concentrations making it impossible to hold very high concentrations for any substantial length of time. Here we report evidence that AA derivates are antiproliferative and cytotoxic molecules at an IC50 lower than AA itself. Among these new molecules, we selected K873 that has cytotoxic and antiproliferative effects on different human tumor cells at tenth micromolar concentration. In a further step, we demonstrated that K873 selectively to kills only cancer cells without being toxic for normal non-dividing (or poorly dividing) cells. Finally, we tested the effect of treatment with K873 (5-10 mg/kg/d by i.p. route) on tumor progression in xenografted immunodeficient mice (BALB/c Nude). Our data suggest that K873 administration strongly inhibits tumor progression. In a previous study using microarrays, we demonstrated that AA decreases the expression of two genes families involved in cell cycle progression, i.e. initiation factor of translation and tRNA synthetases. Here we show that K873 treatment also decreases the expression of four of these genes in xenografted tumors, in proportions similar to that previously observed with AA. Taken together, our data suggest that AA and K873 share similar action. Our findings suggest that AA derivatives could be a promising new class of anti-cancer drugs, either alone or in combination with other molecules.