Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, which are CLPP activators, which inhibit OXPHOS indirectly and have demonstrated safety in patients. METHODS: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201 and ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic efficacy in vivo in UM liver metastasis models. RESULTS: CLPP expression was detected in primary and mUM patient samples. ONC201 and 212 decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis in human UM cell lines in vitro. ONC212 inhibited OXPHOS, increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. CONCLUSIONS: Imipridones are a promising strategy for further testing and development in mUM.

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

A chirality-dependent action of vitamin C in suppressing Kirsten rat sarcoma mutant tumor growth by the oxidative combination: Rationale for cancer therapeutics.

Kirsten rat sarcoma (KRAS) mutant cancers, which constitute the vast majority of pancreatic tumors, are characterized by their resistance to established therapies and high mortality rates. Here, we developed a novel and extremely effective combinational therapeutic approach to target KRAS mutant tumors through the generation of a cytotoxic oxidative stress. At high concentrations, vitamin C (VC) is known to provoke oxidative stress and selectively kill KRAS mutant cancer cells, although its effects are limited when it is given as monotherapy. We found that the combination of VC and the oxidizing drug arsenic trioxide (ATO) is an effective therapeutic treatment modality. Remarkably, its efficiency is dependent on chirality of VC as its enantiomer d-optical isomer of VC (d-VC) is significantly more potent than the natural l-optical isomer of VC. Thus, our results demonstrate that the oxidizing combination of ATO and d-VC is a promising approach for the treatment of KRAS mutant human cancers.

It Is Time to Re-Evaluate the Peer Review Process for Preclinical Research.

Problems in peer review, the backbone of maintaining high standards in scientific publishing, have led to wide spread discontent within the scientific community. Training in the peer review process and a simpler format to assist in decision making are possible courses to improve and expedite the process of peer review and scientific publishing.

Intracrine VEGF signalling mediates colorectal cancer cell migration and invasion.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are key regulators of angiogenesis, affecting endothelial cell survival and function. However, the effect of VEGF-VEGFR signalling on tumour cell function is not well understood. Our previous studies in colorectal cancer (CRC) cells have demonstrated an intracrine VEGF/VEGFR1 signalling mechanism that mediates CRC cell survival and chemo-sensitivity. Since extracellular VEGF signalling regulates migration of endothelial cells and various tumour cells, we attempted to determine whether intracrine VEGF signalling affects CRC cell motility. METHODS: Migration and invasion of CRC cells, with and without VEGF or VEGFR1 depletion, were assayed using transwell migration chambers. Changes in cell morphology, epithelial-mesenchymal transition (EMT) markers, and markers of cell motility were assessed by immunostaining and western blot. RESULTS: Depletion of intracellular VEGF and VEGFR1 in multiple CRC cell lines led to strong inhibition of migration and invasion of CRC cells. Except for Twist, there were no significant differences in markers of EMT between control and VEGF/VEGFR1-depleted CRC cells. However, VEGF/VEGFR1-depleted CRC cells demonstrated a significant reduction in levels of phosphorylated focal adhesion kinase and its upstream regulators pcMET and pEGFR. CONCLUSIONS: Inhibition of intracrine VEGF signalling strongly inhibits CRC cell migration and invasion by regulating proteins involved in cell motility.

Enhancing efficacy of the MEK inhibitor trametinib with paclitaxel in KRAS -mutated colorectal cancer.

Background: KRAS is frequently mutated in the tumors of patients with metastatic colorectal cancer (mCRC) and thus represents a valid target for therapy. However, the strategies of targeting KRAS directly and targeting the downstream effector mitogen-activated protein kinase kinase (MEK) via monotherapies have shown limited efficacy. Thus, there is a strong need for novel, effective combination therapies to improve MEK-inhibitor efficacy in patients with KRAS -mutated mCRC. Objective: Our objective was to identify novel drug combinations that enhance MEK-inhibitor efficacy in patients with KRAS -mutated mCRC. Design: In this study, we performed unbiased high-throughput screening (HTS) to identify drugs that enhance the efficacy of MEK inhibitors in vitro , and we validated the efficacy of the drugs in vivo . Methods: HTS was performed using 3-dimensional CRC spheroids. Trametinib, the anchor drug, was probed with 2 clinically ready libraries of 252 drugs to identify effective drug combinations. The effects of the drug combinations on CRC cell proliferation and apoptosis were further validated using cell growth assays, flow cytometry, and biochemical assays. Proteomic and immunostaining studies were performed to determine the effects of the drugs on molecular signaling and cell division. The effects of the drug combinations were examined in vivo using CRC patient-derived xenografts. Results: HTS identified paclitaxel as being synergistic with trametinib. In vitro validation showed that, compared with monotherapies, this drug combination demonstrated strong inhibition of cell growth, reduced colony formation, and enhanced apoptosis in multiple KRAS -mutated CRC cell lines. Mechanistically, combining trametinib with paclitaxel led to alterations in signaling mediators that block cell cycle progression and increases in microtubule stability that resulted in significantly higher defects in the mitosis. Finally, the combination of trametinib with paclitaxel exhibited significant inhibition of tumor growth in several KRAS -mutant patient-derived xenograft mouse models. Conclusion: Our data provide evidence supporting clinical trials of trametinib with paclitaxel as a novel therapeutic option for patients with KRAS -mutated, metastatic CRC.

Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

Purpose: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, CLPP activators which reduce OXPHOS indirectly and have demonstrated safety in patients. Experimental Design: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201, ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic effects in vivo in UM liver metastasis models. Results: CLPP expression was confirmed in primary and mUM patient samples. ONC201/212 treatment of UM cell lines in vitro decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis. ONC212 increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. Conclusion: Imipridones are a promising strategy for further testing and development in mUM.

Combining MEK and SRC inhibitors for treatment of colorectal cancer demonstrate increased efficacy in vitro but not in vivo.

Metastatic colorectal cancer (mCRC) is the second leading cause of cancer deaths in the United States. More than 50% of patients with mCRC harbor mutations of the oncogenic driver RAS (KRAS or NRAS). Because directly targeting most mutations of RAS is technically challenging, researchers have concentrated on targeting MEK, a downstream mediator of RAS. However, targeting MEK as single-agent therapy is ineffective in patients with mCRC. We hypothesize that combining a MEK inhibitor with other agents can enhance the efficacy of MEK targeting in mCRC. Unbiased high-throughput screening (HTS) was performed to identify drugs that enhance the efficacy of MEK inhibitors. HTS was performed with KRAS-mutated CRC cells using the MEK inhibitor trametinib as a "backbone" and two "clinically ready" compound libraries approved by the U.S. Food and Drug Administration or in clinical trials. HTS demonstrated that the combination of the SRC inhibitor dasatinib and trametinib was synergistic in CRC cells in vitro (MTT and colony formation assays). Analysis of markers for cell proliferation and apoptosis using fluorescence-activated cell sorting, reverse-phase protein array, or Western blotting demonstrated decreased cell proliferation and increased cell death when targeting both SRC and MEK as compared to single agents in multiple CRC cell lines. However, combining dasatinib and trametinib in vivo at doses in mice equivalent to doses used in humans failed to significantly enhance the antitumor activity of trametinib when compared to that of trametinib alone. These results underscore the importance of performing careful preclinical in vivo validation studies using clinically relevant doses as a prerequisite for translating in vitro findings to the clinic.

Vincristine Enhances the Efficacy of MEK Inhibitors in Preclinical Models of KRAS-mutant Colorectal Cancer.

Mutations in KRAS are found in more than 50% of tumors from patients with metastatic colorectal cancer (mCRC). However, direct targeting of most KRAS mutations is difficult; even the recently developed KRASG12C inhibitors failed to show significant benefit in patients with mCRC. Single agents targeting mitogen-activated protein kinase kinase (MEK), a downstream mediator of RAS, have also been ineffective in colorectal cancer. To identify drugs that can enhance the efficacy of MEK inhibitors, we performed unbiased high-throughput screening using colorectal cancer spheroids. We used trametinib as the anchor drug and examined combinations of trametinib with the NCI-approved Oncology Library version 5. The initial screen, and following focused validation screens, identified vincristine as being strongly synergistic with trametinib. In vitro, the combination strongly inhibited cell growth, reduced clonogenic survival, and enhanced apoptosis compared with monotherapies in multiple KRAS-mutant colorectal cancer cell lines. Furthermore, this combination significantly inhibited tumor growth, reduced cell proliferation, and increased apoptosis in multiple KRAS-mutant patient-derived xenograft mouse models. In vivo studies using drug doses that reflect clinically achievable doses demonstrated that the combination was well tolerated by mice. We further determined that the mechanism underlying the synergistic effect of the combination was due to enhanced intracellular accumulation of vincristine associated with MEK inhibition. The combination also significantly decreased p-mTOR levels in vitro, indicating that it inhibits both RAS-RAF-MEK and PI3K-AKT-mTOR survival pathways. Our data thus provide strong evidence that the combination of trametinib and vincristine represents a novel therapeutic option to be studied in clinical trials for patients with KRAS-mutant mCRC. SIGNIFICANCE: Our unbiased preclinical studies have identified vincristine as an effective combination partner for the MEK inhibitor trametinib and provide a novel therapeutic option to be studied in patients with KRAS-mutant colorectal cancer.

Mitotic centromere-associated kinesin (MCAK) mediates paclitaxel resistance.

Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III ß-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.

Cytospin-A Regulates Colorectal Cancer Cell Division and Migration by Modulating Stability of Microtubules and Actin Filaments.

Proteins that interact with cytoskeletal elements play important roles in cell division and are potentially important targets for therapy in cancer. Cytospin-A (CYTSA), a protein known to interact with actin and microtubules, has been previously described to be important in various developmental disorders, including oblique facial clefting. We hypothesized that CYTSA plays an important role in colorectal cancer (CRC) cell division. The effects of CYTSA depletion on CRC cell proliferation were analyzed using cell growth assays, microscopic analyses of live and fixed cells, and time-lapse imaging. CYTSA depletion led to inhibition of cell proliferation, significant increases in CRC cell death, and accumulation of doublet cells during and following cell division. Depletion of CYTSA also resulted in strong inhibition of CRC cell migration and invasion. Mechanistically, CYTSA depletion resulted in significant decreases in the stability of microtubules and altered polymerization of actin filaments in CRC cells. Finally, bioinformatic analyses were performed to determine the correlation between CYTSA expression and survival of patients with CRC. Interestingly, a strong correlation between high CYTSA expression and poor survival was observed in the TCGA adenocarcinoma data set but not in an independent data set. Since inhibiting CYTSA significantly reduces CRC cell proliferation, migration, and invasion, targeting CYTSA may be a potential novel therapeutic option for patients with metastatic CRC.

Targeting IRS-1/2 in Uveal Melanoma Inhibits In Vitro Cell Growth, Survival and Migration, and In Vivo Tumor Growth.

Uveal melanoma originating in the eye and metastasizing to the liver is associated with poor prognosis and has only one approved therapeutic option. We hypothesized that liver-borne growth factors may contribute to UM growth. Therefore, we investigated the role of IGF-1/IGF-1R signaling in UM. Here, we found that IRS-1, the insulin receptor substrate, is overexpressed in both UM cells and tumors. Since we previously observed that IGF-1R antibody therapy was not clinically effective in UM, we investigated the potential of NT157, a small molecule inhibitor of IRS-1/2, in blocking this pathway in UM. NT157 treatment of multiple UM cell lines resulted in reduced cell growth and migration and increased apoptosis. This treatment also significantly inhibited UM tumor growth in vivo, in the chicken egg chorioallantoic membrane (CAM) and subcutaneous mouse models, validating the in vitro effect. Mechanistically, through reverse phase protein array (RPPA), we identified significant proteomic changes in the PI3K/AKT pathway, a downstream mediator of IGF-1 signaling, with NT157 treatment. Together, these results suggest that NT157 inhibits cell growth, survival, and migration in vitro, and tumor growth in vivo via inhibiting IGF-1 signaling in UM.

Liver Endothelium Promotes HER3-Mediated Cell Survival in Colorectal Cancer with Wild-Type and Mutant KRAS.

We previously identified that human epidermal growth factor receptor 3 (HER3, also known as ERBB3) is a key mediator in liver endothelial cell (EC) promoting colorectal cancer growth and chemoresistance, and suggested HER3-targeted therapy as a strategy for treating patients with metastatic colorectal cancer in the liver. Meanwhile, KRAS mutations occur in 40%-50% of metastatic colorectal cancer and render colorectal cancer resistant to therapies targeting the other HER family protein epidermal growth factor receptor (EGFR). It is necessary to elucidate the roles of KRAS mutation status in HER3-mediated cell survival and colorectal cancer response to HER3 inhibition. In the present study, we used primary ECs isolated from non-neoplastic liver tissues to recapitulate the liver EC microenvironment. We demonstrated that liver EC-secreted factors activated colorectal cancer-associated HER3, and increased colorectal cancer cell survival in vitro and promoted colorectal cancer patient-derived xenograft tumor growth in vivo. Moreover, we determined that blocking HER3, either by siRNA knockdown or the humanized antibody seribantumab, blocked EC-induced colorectal cancer survival in vitro in both KRAS wild-type and mutant colorectal cancer cells, and the HER3 antibody seribantumab significantly decreased colorectal cancer tumor growth and sensitized tumors to chemotherapy in an orthotopic xenograft model with colorectal cancer tumors developed in the liver. In summary, our findings demonstrated that blocking HER3 had significant effects on attenuating liver EC-induced colorectal cancer cell survival independent of the KRAS mutation status. IMPLICATIONS: This body of work highlighted a potential strategy of using HER3 antibodies in combination with standard chemotherapy agents for treating patients with either KRAS wild-type or KRAS mutant metastatic colorectal cancer.

A minor beta-tubulin essential for mammalian cell proliferation.

Mammals use tubulin from multiple genes to construct microtubules. Some genes are expressed in a tissue specific manner, while others are expressed in almost all cell types. beta5-Tubulin is a minor, ubiquitous isoform whose overexpression was recently shown to disrupt microtubules. Using inhibitory RNA, we now report that suppression of beta5 production in both human and hamster cells blocks cell proliferation. Cells depleted of beta5 either trigger the mitotic checkpoint and undergo apoptosis; or they experience a transient mitotic block, a high incidence of lagging chromosomes, and progression into G1 without cytokinesis to become large, flat cells with elevated DNA content. Microtubules appear to be normally organized in cells depleted of beta5, but they are rich in acetylated alpha-tubulin indicating that they may be more stable than normal. The results provide the first evidence that a specific isoform of beta-tubulin is required for mitosis.

Endothelial Cells Promote Colorectal Cancer Cell Survival by Activating the HER3-AKT Pathway in a Paracrine Fashion.

The regulation of colorectal cancer cell survival pathways remains to be elucidated. Previously, it was demonstrated that endothelial cells (EC) from the liver (liver parenchymal ECs or LPEC), the most common site of colorectal cancer metastases, secrete soluble factors in the conditioned medium (CM) that, in turn, increase the cancer stem cell phenotype in colorectal cancer cells. However, the paracrine effects of LPECs on other colorectal cancer cellular functions have not been investigated. Here, results showed that CM from LPECs increased cell growth and chemoresistance by activating AKT in colorectal cancer cells in vitro. Using an unbiased receptor tyrosine kinase array, it was determined that human epidermal growth factor receptor 3 (ERBB3/HER3) was activated by CM from LPECs, and it mediated AKT activation, cell growth, and chemoresistance in colorectal cancer cells. Inhibition of HER3, either by an inhibitor AZD8931 or an antibody MM-121, blocked LPEC-induced HER3-AKT activation and cell survival in colorectal cancer cells. In addition, CM from LPECs increased in vivo tumor growth in a xenograft mouse model. Furthermore, inhibiting HER3 with AZD8931 significantly blocked tumor growth induced by EC CM. These results demonstrated a paracrine role of liver ECs in promoting cell growth and chemoresistance via activating HER3-AKT in colorectal cancer cells. IMPLICATIONS: This study suggested a potential of treating patients with metastatic colorectal cancer with HER3 antibodies/inhibitors that are currently being assessed in clinical trials for various cancer types.

A Survey on Data Reproducibility and the Effect of Publication Process on the Ethical Reporting of Laboratory Research.

Purpose: The successful translation of laboratory research into effective therapies is dependent upon the validity of peer-reviewed publications. However, several publications in recent years suggested that published scientific findings could be reproduced only 11% to 45% of the time. Multiple surveys attempted to elucidate the fundamental causes of data irreproducibility and underscored potential solutions, more robust experimental designs, better statistics, and better mentorship. However, no prior survey has addressed the role of the review and publication process on honest reporting.Experimental Design: We developed an anonymous online survey intended for trainees involved in bench research. The survey included questions related to mentoring/career development, research practice, integrity, and transparency, and how the pressure to publish and the publication process itself influence their reporting practices.Results: Responses to questions related to mentoring and training practices were largely positive, although an average of approximately 25% did not seem to receive optimal mentoring. A total of 39.2% revealed having been pressured by a principle investigator or collaborator to produce "positive" data. About 62.8% admitted that the pressure to publish influences the way they report data. The majority of respondents did not believe that extensive revisions significantly improved the manuscript while adding to the cost and time invested.Conclusions: This survey indicates that trainees believe that the pressure to publish affects honest reporting, mostly emanating from our system of rewards and advancement. The publication process itself affects faculty and trainees and appears to influence a shift in their ethics from honest reporting ("negative data") to selective reporting, data falsification, or even fabrication. Clin Cancer Res; 24(14); 3447-55. ©2018 AACR.

Macrophage conditioned medium promotes colorectal cancer stem cell phenotype via the hedgehog signaling pathway.

BACKGROUND: There is conflicting data on the role of macrophages in colorectal cancer (CRC); some studies have shown that macrophages can exert an anti-tumor effect whereas others show that macrophages are tumor promoting. We sought to determine the role of conditioned medium (CM) from macrophages, in particular classically activated macrophages, on the development of the CSC phenotype in CRC cells, which is believed to mediate tumor growth and chemoresistance. METHODS: Murine (CT26) and human (HCP-1) CRC cell lines were treated with CM from lipopolysaccharide (LPS)-activated murine macrophages. The CSC population was assessed using the sphere-forming assay and aldehyde dehydrogenase assay. Chemoresistance studies were performed using the MTT assay. CSC transcription factors and SHH protein were analyzed by Western blotting. RESULTS: The results showed that LPS-activated macrophage CM induced the CSC phenotype in CRC cells. Further studies showed that the CSC phenotype was mediated by the sonic hedgehog (SHH)-Gli signaling pathway, which is known to drive self-renewal; these effects were blocked by depletion of SHH in macrophage CM. In addition, LPS-activated macrophage CM enhanced chemoresistance. CONCLUSIONS: LPS-activated macrophages play an active role in promoting the CSC phenotype through activation of the SHH-Gli signaling pathway in CRC cells.

The G23 and G25 genes of temperate mycobacteriophage L1 are essential for the transcription of its late genes.

Two lysis-defective but DNA synthesis non-defective temperature-sensitive (ts) mutants of mycobacteriophage L1, L1G23ts23 and L1G25ts889 were found to be defective also in phage-specific RNA synthesis in the late period of their growth at 42 degrees C, each to the extent of 50% of that at 32 degrees C. The double mutant, L1G23ts23G25ts889 showed the ts defect in phage RNA synthesis that was nearly additive of those shown individually by the two single-mutant parents. Both G23 and G25 were shown to start functioning sometimes between 30 and 45 min after infection but the former gene might be dispensable after 45 min, while the latter was not. Northern analysis also shows that at 42 degrees C, L1G23ts23 affects RNA synthesis more strongly than L1G25ts889 from L1 DNA segments that serve as the template for late gene transcription. Among the 21 virion and 12 non-virion late proteins synthesized by L1, L1G23ts23 is defective in the synthesis of at least 9 virion and all of non-virion proteins at 42 degrees C. In contrast, L1G25ts889 is completely defective in synthesis of all the 33 late proteins. Possible roles of G23 and G25 in the positive regulation of transcription of different sets of late genes of L1 have been discussed.

A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation.

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.

Endothelial cells activate the cancer stem cell-associated NANOGP8 pathway in colorectal cancer cells in a paracrine fashion.

In colorectal cancer (CRC), cancer stem cells (CSCs) have been hypothesized to mediate cell survival and chemoresistance. Previous studies from our laboratory described a role for liver parenchymal endothelial cells (LPECs) in mediating the CSC phenotype in CRC cells in a paracrine/angiocrine fashion. The objectives of this study were to determine whether endothelial cells (ECs) from different organs can induce the CSC phenotype in CRC cells and to elucidate the signaling pathways involved. We treated a newly developed CRC cell line (HCP-1) and established CRC cell lines (HT29 and SW480) with conditioned medium (CM) from primary ECs isolated from nonmalignant liver, lung, colon mucosa, and kidney. Our results showed that CM from ECs from all organs increased the number of CSCs, as determined by sphere formation, and protein levels of NANOG and OCT4 in CRC cells. With the focus of further elucidating the role of the liver vascular network in mediating the CSC phenotype, we demonstrated that CM from LPECs increased resistance to 5-fluorouracil in CRC cells. Moreover, we showed that LPEC CM specifically induced NANOGP8 expression in CRC cells by specific enzyme digestion and a luciferase reporter assay using a vector containing the NANOGP8 promoter. Lastly, we found that LPEC CM-induced NANOGP8 expression and sphere formation were mediated by AKT activation. Our studies demonstrated a paracrine role for ECs in regulating the CSC phenotype and chemoresistance in CRC cells by AKT-mediated induction of NANOGP8. These studies suggest a more specific approach to target CSCs by blocking the expression of NANOGP8 in cancer cells.