Anne-Marie Bleau

Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer

BackgroundMortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312.ResultsThe aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo.ConclusionsWe conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.

Epithelial to mesenchymal transition and cancer stem cell phenotypes leading to liver metastasis are abrogated by the novel TGFβ1-targeting peptides P17 and P144.

Colorectal cancer (CRC) frequently metastasizes to the liver, a phenomenon that involves the participation of transforming-growth-factor-β(1) (TGFβ(1)). Blockade of the protumorigenic effects elicited by TGFβ(1) in advanced CRC could attenuate liver metastasis. We aimed in the present study to assess the antimetastatic effect of TGFβ(1)-blocking peptides P17 and P144, and to study mechanisms responsible for this activity in a mouse model. Colon adenocarcinoma cells expressing luciferase were pretreated with TGFβ(1) (Mc38-luc(TGFβ1) cells), injected into the spleen of mice and monitored for tumor development. TGFβ(1) increased primary tumor growth and liver metastasis, whereas systemic treatment of mice with either P17 or P144 significantly reduced tumor burden (p<0.01). In metastatic nodules, mitotic/apoptotic ratio, mesenchymal traits and angiogenesis (evaluated by CD-31, as well as circulating endothelial and progenitor cells) induced by TGFβ(1) were consistently reduced following injection of peptides. In vitro experiments revealed a direct effect of TGFβ(1) in Mc38 cells, which resulted in activation of Smad2, Smad3 and Smad1/5/8, and increased invasion and transendothelial migration, whereas blockade of TGFβ(1)-signaling reverted these features. Because TGFβ(1)-mediated epithelial-mesenchymal transition (EMT) has been suggested to induce a cancer stem cell (CSC) phenotype, we analyzed the ability of this cytokine to induce tumorsphere formation and the expression of CSC markers. In TGFβ(1)-treated cells, tumorspheres were enriched in CD44 and SOX2, which were diminished in the presence of P17. Our data provide a preclinical rationale to evaluate P17 and P144 as potential therapeutic options for the treatment of metastatic CRC.

Metastatic dormancy: a complex network between cancer stem cells and their microenvironment.

Metastasis represents the major threat of cancer progression and generally emerges years after the detection of the primary tumor. An important rate-limiting step resides in cellular dormancy, where a disseminated tumor cell remains in a quiescent state at a remote organ. Herein we review the molecular mechanisms leading to tumor dormancy, mainly in regards to cellular quiescence and the tumor microenvironment. Based on the current published literature, we provide evidence that links the cancer stem cell (CSC) theory with dormancy and metastasis. Once a disseminated tumor cell reaches a target tissue, a tight regulation imposed by the foreign microenvironment will dictate the fate of these cells, which implies a balance in the secretion of soluble factors, modulation of the extracellular matrix and the angiogenic switch. We investigate thoroughly whether the CSC theory could also apply to metastasis initiation. In fact, the resistance of CSCs to therapy, leading to the minimal residual disease and cellular quiescence phenotypes, predisposes for the development of metastases. Finally, we describe the new technologies available for the identification of circulating tumor cells (CTCs), as well as their clinical relevance in dormancy of metastatic cancer patients.

Co-migration of colon cancer cells and CAFs induced by TGFβ 1 enhances liver metastasis

Colorectal cancer (CRC) cells often metastatize to the liver. Cancer-associated fibroblasts (CAFs) enhance metastasis by providing cytokines that create a favorable microenvironment and by inducing co-dissemination with tumor cells. However, the mechanisms of co-metastatization remain elusive. The aim of this study is to assess the role of TGFβ1 in CRC cell–CAFs attachment and its impact on liver metastasis. CAFs were obtained after xenotransplantation of Mc38 cells into EGFP-C57BL/6 mice. Attachment experiments with CRC cells and CAFs (with or without TGFβ1 and the inhibitory peptide P17) were carried out, as well as in vivo liver metastasis assays. TGFβ1 induced adhesion of CRC cells to CAFs, whereas exposure to P17 abrogated this effect. Co-injection of Mc38 cells with CAFs intrasplenically increased liver metastasis, as compared to injection of tumor cells alone. Pretreatment of Mc38 cells with TGFβ1 enhanced the metastatic burden, in comparison to untreated Mc38 + CAFs. TGFβ1-pretreated Mc38 cells co-metastatized with CAFs to the liver in a highly efficient way. Importantly, the metastatic burden was significantly reduced (p < 0.001) when P17 was administered in mice. The number of PCNA+ and CD-31+ cells was also reduced by P17 in these animals, indicating a decrease in proliferation and angiogenesis upon TGFβ1 signaling blockade. Through microarray analysis, we identified potential TGFβ1-regulated genes that may mediate cancer cell–stroma interactions to increase metastasis. In conclusion, TGFβ1 promotes co-travelling of CRC cells and CAFs to the liver to enhance metastasis. Our results strongly support the use of TGFβ1 targeted drugs as a novel strategy to reduce liver metastasis in CRC patients.

The inhibitor of differentiation isoform Id1b, generated by alternative splicing, maintains cell quiescence and confers self-renewal and cancer stem cell-like properties.

Id1 has been shown to play a critical role in tumorigenesis and angiogenesis. Moreover, recent reports have involved Id1 in the maintenance of cancer stem cell features in some tumor types. The Id1 gene generates two isoforms through alternative splicing: Id1a and Id1b. We have investigated the role of each isoform in cancer development. Using lentiviral systems we modified the endogenous expression of each of these isoforms in cancer cells and analyzed their biological effect both in vitro and in vivo. Overexpression of Id1b in murine CT26 and 3LL cells caused a G0/G1 cell cycle arrest and reduced proliferation, clonogenicity and phospho-ERK1/2 levels, while increasing p27 levels. High levels of Id1a had an opposite effect and the proportion of cells in the S phase increased significantly. In vivo models confirmed the inhibitory role of Id1b in primary tumor growth and metastasis. Through microarray analysis we found that the cancer stem cell (CSC) markers ALDH1A1 and Notch-1 were up-regulated specifically in Id1b-overexpressing cells. By using qPCR we also found overexpression of Sca-1, Tert, Sox-2 and Oct-4 in these cells. Increased levels of Id1b promoted self-renewal and CSC-like properties, as shown by their high capacity for developing secondary tumorspheres and retaining the PKH26 dye. The acquisition of CSC phenotype was confirmed in human PC-3 cells that overexpressed Id1b. Our results show that Id1b maintains cells in a quiescent state and promotes self-renewal and CSC-like features. On the contrary, Id1a promotes cell proliferation.

New syngeneic inflammatory‐related lung cancer metastatic model harboring double KRAS/WWOX alterations

New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein, we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb‐c mice. Tumor cell lines were obtained from a silica‐mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N‐nitrosodimethylamine, a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas, and their intracardiac injection led to prominent colonization of various organs (bone, lung, liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G–A transition), accompanied by a homozygous deletion of the WW domain‐containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5–8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity, confirming the tumor suppressor function of this gene in this particular model. Interestingly, we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non‐small‐cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases, with significant reduction in protein levels as compared to normal tissues. Overall, our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb‐c mice background and highlights the importance of WWOX deletion in lung carcinogenesis.

Sphere-derived tumor cells exhibit impaired metastasis by a host-mediated quiescent phenotype

The spread of lung cancer cells to distant sites represents a common event associated with poor prognosis. A fraction of tumor cells named cancer stem cells (CSCs) have the ability to overcome therapeutic stress and remain quiescent. However, whether these CSCs have also the capacity to initiate and sustain metastasis remains unclear. Here, we used tumor sphere cultures (TSC) isolated from mouse and human lung cancer models to enrich for CSCs, and assessed their metastatic potential as compared to non-CSCs. As expected, TSC overexpressed a variety of stem cell markers and displayed chemoresistance. The CSC phenotype of TSC was confirmed by their higher growth ability in soft agar and tumorigenic potential in vivo, despite their reduced in vitro cell growth kinetics. Surprisingly, the appearance of spontaneous lung metastases was strongly delayed in mice injected with TSC as compared to non-TSC cells. Similarly, this finding was confirmed in several other models of metastasis, an effect associated with a retarded colonization activity. Interestingly, such delay correlated with a quiescent phenotype whose underlined mechanisms included an increase in p27 protein and lower phospho-ERK1/2 levels. Thus, these data suggest that cells enriched for CSC properties display an impaired metastatic activity, a finding with potential clinical implications.

Tivanisiran, a novel siRNA for the treatment of dry eye disease

ABSTRACT Introduction: Dry eye disease (DED) is characterized by an alteration of the tear film with ocular inflammation and neurosensory abnormalities. The main clinical signs of this condition are tear instability and ocular damage. Although DED has gained significant attention in the past few years, limited prescription treatment options are available for patients. Areas covered: The current manuscript summarizes the pre-clinical and clinical development of tivanisiran, a novel small interfering oligonucleotide of RNA (siRNA) used for the treatment of DED. Tivanisiran was designed to silence Transient Receptor Potential Vanilloid 1 (TRPV1); herein the chemistry and mechanism of action of this new compound is also described. Expert opinion: Drugs currently on the market mostly target the inflammatory component of the disease and show only partial efficacy. New compounds addressing other aspects of the disease would provide significant advantages and contribute to a more personalized treatment of the disease. Tivanisiran has been designed to reduce ocular discomfort and pain, and was shown to improve ocular hyperemia and tear quality in human and animal models. Consequently, if the results of the ongoing and future clinical trials meet their study endpoints, tivanisiran could be submitted to obtain approval for the treatment of DED.

Managing Intraocular Pressure: Innovation in Glaucoma Management

Primary open-angle glaucoma is a progressive ocular neuropathy that if left untreated may lead to blindness. The main risk factor for developing glaucoma is increased intraocular pressure. Intraocular pressure is regulated by the balance of aqueous humour synthesis and secretion into the eye and outflow from the eye; therefore, most therapies for glaucoma seek lowering intraocular pressure to avoid disease progression. There are several types of drugs in the market for the treatment of glaucoma, but there are still unmet needs to be overcome; therefore, significant effort has been put in the last few years to develop new medicines with innovative mechanisms of action as well as devices to improve quality of life in glaucoma patients. The present review offers a thorough revision of the latest advances in the glaucoma therapy field, focusing on innovative approaches, new targets and new mechanisms of action.