Replication Study: Melanoma genome sequencing reveals frequent PREX2 mutations

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Chroscinski et al., 2014) that described how we intended to replicate selected experiments from the paper "Melanoma genome sequencing reveals frequent PREX2 mutations" (Berger et al., 2012). Here we report the results of those experiments. We regenerated cells stably expressing ectopic wild-type and mutant phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2 (PREX2) using the same immortalized human NRASG12D melanocytes as the original study. Evaluation of PREX2 expression in these newly generated stable cells revealed varying levels of expression among the PREX2 isoforms, which was also observed in the stable cells made in the original study (Figure S6A; Berger et al., 2012). Additionally, ectopically expressed PREX2 was found to be at least 5 times above endogenous PREX2 expression. The monitoring of tumor formation of these stable cells in vivo resulted in no statistically significant difference in tumor-free survival driven by PREX2 variants, whereas the original study reported that these PREX2 mutations increased the rate of tumor incidence compared to controls (Figure 3B and S6B; Berger et al., 2012). Surprisingly, the median tumor-free survival was 1 week in this replication attempt, while 70% of the control mice were reported to be tumor-free after 9 weeks in the original study. The rapid tumor onset observed in this replication attempt, compared to the original study, makes the detection of accelerated tumor growth in PREX2 expressing NRASG12D melanocytes extremely difficult. Finally, we report meta-analyses for each result.

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Replication Study: Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs

eLife ◽

10.7554/elife.17584 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 34

Author(s):

Christine Mantis ◽

Irawati Kandela ◽

Fraser Aird ◽

Keyword(s):

Cancer Biology ◽

Xenograft Model ◽

Original Study ◽

Cancer Drug ◽

Tunel Staining ◽

Positive Staining ◽

Cancer Drugs ◽

Tumor Weight ◽

Significant Difference ◽

Meta Analyses

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Kandela et al., 2015) that described how we intended to replicate selected experiments from the paper “Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs“ (Sugahara et al., 2010). Here we report the results of those experiments. We found that coadministration with iRGD peptide did not have an impact on permeability of the chemotherapeutic agent doxorubicin (DOX) in a xenograft model of prostate cancer, whereas the original study reported that it increased the penetrance of this cancer drug (Figure 2B; Sugahara et al., 2010). Further, in mice bearing orthotopic 22Rv1 human prostate tumors, we did not find a statistically significant difference in tumor weight for mice treated with DOX and iRGD compared to DOX alone, whereas the original study reported a decrease in tumor weight when DOX was coadministered with iRGD (Figure 2C; Sugahara et al., 2010). In addition, we did not find a statistically significant difference in TUNEL staining in tumor tissue between mice treated with DOX and iRGD compared to DOX alone, while the original study reported an increase in TUNEL positive staining with iRGD coadministration (Figure 2D; Sugahara et al., 2010). Similar to the original study (Supplemental Figure 9A; Sugahara et al., 2010), we did not observe an impact on mouse body weight with DOX and iRGD treatment. Finally, we report meta-analyses for each result.

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Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

eLife ◽

10.7554/elife.25306 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 8

Author(s):

Xiaochuan Shan ◽

Juan Jose Fung ◽

Alan Kosaka ◽

Gwenn Danet-Desnoyers ◽

Keyword(s):

Growth Inhibition ◽

Effective Treatment ◽

Cell Line ◽

Cancer Biology ◽

Vehicle Control ◽

Original Study ◽

Selective Growth ◽

Significant Difference ◽

K 562 Cells ◽

Meta Analyses

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al., 2011). Here, we report the results of those experiments. We found treatment of MLL-fusion leukaemia cells (MV4;11 cell line) with the BET bromodomain inhibitor I-BET151 resulted in selective growth inhibition, whereas treatment of leukaemia cells harboring a different oncogenic driver (K-562 cell line) did not result in selective growth inhibition; this is similar to the findings reported in the original study (Figure 2A and Supplementary Figure 11A,B; Dawson et al., 2011). Further, I-BET151 resulted in a statistically significant decrease in BCL2 expression in MV4;11 cells, but not in K-562 cells; again this is similar to the findings reported in the original study (Figure 3D; Dawson et al., 2011). We did not find a statistically significant difference in survival when testing I-BET151 efficacy in a disseminated xenograft MLL mouse model, whereas the original study reported increased survival in I-BET151 treated mice compared to vehicle control (Figure 4B,D; Dawson et al., 2011). Differences between the original study and this replication attempt, such as different conditioning regimens and I-BET151 doses, are factors that might have influenced the outcome. We also found I-BET151 treatment resulted in a lower median disease burden compared to vehicle control in all tissues analyzed, similar to the example reported in the original study (Supplementary Figure 16A; Dawson et al., 2011). Finally, we report meta-analyses for each result.

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Replication Study: Wnt activity defines colon cancer stem cells and is regulated by the microenvironment

eLife ◽

10.7554/elife.45426 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 1

Author(s):

Anthony Essex ◽

Javier Pineda ◽

Grishma Acharya ◽

Hong Xin ◽

James Evans ◽

...

Keyword(s):

Stem Cells ◽

Colon Cancer ◽

Cancer Stem Cells ◽

Cancer Biology ◽

Statistical Significance ◽

Original Study ◽

Secreted Factors ◽

Colon Cancer Stem Cells ◽

Meta Analyses

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Evans et al., 2015), that described how we intended to replicate selected experiments from the paper ‘Wnt activity defines colon cancer stem cells and is regulated by the microenvironment’ (Vermeulen et al., 2010). Here, we report the results. Using three independent primary spheroidal colon cancer cultures that expressed a Wnt reporter construct we observed high Wnt activity was associated with the cell surface markers CD133, CD166, and CD29, but not CD24 and CD44, while the original study found all five markers were correlated with high Wnt activity (Figure 2F; Vermeulen et al., 2010). Clonogenicity was highest in cells with high Wnt activity and clonogenic potential of cells with low Wnt activity were increased by myofibroblast-secreted factors, including HGF. While the effects were in the same direction as the original study (Figure 6D; Vermeulen et al., 2010) whether statistical significance was reached among the different conditions varied. When tested in vivo, we did not find a difference in tumorigenicity between high and low Wnt activity, while the original study found cells with high Wnt activity were more effective in inducing tumors (Figure 7E; Vermeulen et al., 2010). Tumorigenicity, however, was increased with myofibroblast-secreted factors, which was in the same direction as the original study (Figure 7E; Vermeulen et al., 2010), but not statistically significant. Finally, we report meta-analyses for each results where possible.

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Replication Study: The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44

eLife ◽

10.7554/elife.43511 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

Author(s):

Xuefei Yan ◽

Beibei Tang ◽

Biao Chen ◽

Yongli Shan ◽

Huajun Yang ◽

...

Keyword(s):

Prostate Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

Binding Sites ◽

Cancer Biology ◽

Original Study ◽

Cd44 Expression ◽

Prostate Cancer Stem Cells ◽

Significant Difference

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Li et al., 2015), that described how we intended to replicate selected experiments from the paper ‘The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44’ (Liu et al., 2011). Here we report the results. We found the microRNA, miR-34a, was expressed at twice the level in CD44+ prostate cancer cells purified from xenograft tumors (LAPC4 cells) compared to CD44- LAPC4 cells, whereas the original study reported miR-34a was underexpressed in CD44+ LAPC4 cells (Figure 1B; Liu et al., 2011). When LAPC4 cells engineered to express miR-34a were injected into mice, we did not observe changes in tumor growth or CD44 expression; however, unexpectedly miR-34a expression was lost in vivo. In the original study, LAPC4 cells expressing miR-34a had a statistically significant reduction in tumor regeneration and reduced CD44 expression compared to control (Figure 4A and Supplemental Figures 4A,B and 5C; Liu et al., 2011). Furthermore, when we tested if miR-34a regulated CD44 through binding sites in the 3’UTR we did not find a statistically significant difference, whereas the original study reported miR-34a decreased CD44 expression that was partially abrogated by mutation of the binding sites in the CD44 3’UTR (Figure 4D; Liu et al., 2011). Finally, where possible, we report meta-analyses for each result.

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Distinct Tumor Microenvironments Are a Defining Feature of Strain-Specific CRISPR/Cas9-Induced MPNSTs

Genes ◽

10.3390/genes11050583 ◽

2020 ◽

Vol 11 (5) ◽

pp. 583 ◽

Cited By ~ 2

Author(s):

Amanda Scherer ◽

Victoria R. Stephens ◽

Gavin R. McGivney ◽

Wade R. Gutierrez ◽

Emily A. Laverty ◽

...

Keyword(s):

Tumor Microenvironment ◽

Mouse Models ◽

Cancer Biology ◽

Myeloid Cell ◽

Inbred Strains ◽

Peripheral Nerve Sheath Tumors ◽

Tumor Onset ◽

The Impact ◽

Strain Dependent

The tumor microenvironment plays important roles in cancer biology, but genetic backgrounds of mouse models can complicate interpretation of tumor phenotypes. A deeper understanding of strain-dependent influences on the tumor microenvironment of genetically-identical tumors is critical to exploring genotype–phenotype relationships, but these interactions can be difficult to identify using traditional Cre/loxP approaches. Here, we use somatic CRISPR/Cas9 tumorigenesis approaches to determine the impact of mouse background on the biology of genetically-identical malignant peripheral nerve sheath tumors (MPNSTs) in four commonly-used inbred strains. To our knowledge, this is the first study to systematically evaluate the impact of host strain on CRISPR/Cas9-generated mouse models. Our data identify multiple strain-dependent phenotypes, including changes in tumor onset and the immune microenvironment. While BALB/c mice develop MPNSTs earlier than other strains, similar tumor onset is observed in C57BL/6, 129X1 and 129/SvJae mice. Indel pattern analysis demonstrates that indel frequency, type and size are similar across all genetic backgrounds. Gene expression and IHC analysis identify multiple strain-dependent differences in CD4+ T cell infiltration and myeloid cell populations, including M2 macrophages and mast cells. These data highlight important strain-specific phenotypes of genomically-matched MPNSTs that have implications for the design of future studies using similar in vivo gene editing approaches.

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Phenolhexyl Isothiocyanate, a New Histone Deacetylase Inhibitor, Can Prevent Tumor Formation and Inhibit Leukemia Cell Growth In Vivo.

Blood ◽

10.1182/blood.v106.11.4625.4625 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4625-4625

Author(s):

Lulu Lu ◽

Xudong Ma ◽

A. Becklemisheva ◽

J.W. Chaio ◽

Delong Liu

Keyword(s):

Cell Growth ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Chemotherapeutic Agents ◽

Tumor Formation ◽

Control Group ◽

Immunodeficient Mice ◽

Standard Design ◽

Significant Difference

Abstract We have demonstrated that phenolhexyl isothiocyanate (PHI) induces growth arrest and apoptosis in leukemia cells HL-60 through inhibition of the activity of histone deacetylases (HDAC), the enhancement of histone acetylation and activation of p21. In this study, we examined the effects of PHI on the growth of HL-60 leukemic xenograft in immunodeficient mice. The PHI was given to the mice by gavage. The maximum tolerated dose by the mice was determined following standard design. To determine the in vivo effect of PHI on leukemia growth, a sub-MTD dose was given to the control and the treatment groups with 17 mice in each group after injection of 1.0 x 10e6 HL-60 cells per mouse. There was a significant reduction in the incidence of tumor formation (94.1% control group vs 58.5% PHI group, p=0.004). In addition, there was also a significant difference in the tumor size between the two groups. Determined at autopsy, the mean weight of control tumors was 0.8 g vs. the tumors of the experimental group 0.35g, revealing a significant reduction of 44.4% (P<0.03). There were no detectable toxicity as evaluated by body and organ weight, and necropsy examination. The histology of the tumor showed increased apoptotic cell death. Apoptosis in the tumors was further confirmed by the cleavage of poly ADP-ribose polymerase (PARP), the target of proteolysis of caspases that execute apoptosis with Western blot analyses. The results suggest that PHI can prevent tumor formation and inhibit leukemia cell growth in vivo without significant toxicity that is routinely associated with conventional chemotherapeutic agents.

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Replication Study: Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis

eLife ◽

10.7554/elife.45120 ◽

2019 ◽

Vol 8 ◽

Author(s):

Mee Rie Sheen ◽

Jennifer L Fields ◽

Brian Northan ◽

Judith Lacoste ◽

Lay-Hong Ang ◽

...

Keyword(s):

Cancer Cells ◽

Tumor Invasion ◽

Cancer Biology ◽

Original Study ◽

Invasion And Metastasis ◽

Caveolin 1 ◽

Primary Mouse ◽

Metastatic Burden ◽

The Difference

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Fiering et al., 2015) that described how we intended to replicate selected experiments from the paper ‘Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis’ (Goetz et al., 2011). Here we report the results. Primary mouse embryonic fibroblasts (pMEFs) expressing caveolin 1 (Cav1WT) demonstrated increased extracellular matrix remodeling in vitro compared to Cav1 deficient (Cav1KO) pMEFs, similar to the original study (Goetz et al., 2011). In vivo, we found higher levels of intratumoral stroma remodeling, determined by fibronectin fiber orientation, in tumors from cancer cells co-injected with Cav1WT pMEFs compared to cancer cells only or cancer cells plus Cav1KO pMEFs, which were in the same direction as the original study (Supplemental Figure S7C; Goetz et al., 2011), but not statistically significant. Primary tumor growth was similar between conditions, like the original study (Supplemental Figure S7Ca; Goetz et al., 2011). We found metastatic burden was similar between Cav1WT and Cav1KO pMEFs, while the original study found increased metastases with Cav1WT (Figure 7C; Goetz et al., 2011); however, the duration of our in vivo experiments (45 days) were much shorter than in the study by Goetz et al. (2011) (75 days). This makes it difficult to interpret the difference between the studies as it is possible that the cells required more time to manifest the difference between treatments observed by Goetz et al. We also found a statistically significant negative correlation of intratumoral remodeling with metastatic burden, while the original study found a statistically significant positive correlation (Figure 7Cd; Goetz et al., 2011), but again there were differences between the studies in terms of the duration of the metastasis studies and the imaging approaches that could have impacted the outcomes. Finally, we report meta-analyses for each result.

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ASPP2 Haploinsufficiency Promotes Tumor Formation in a Mouse Model.

Blood ◽

10.1182/blood.v108.11.4333.4333 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4333-4333

Author(s):

Kerstin M. Kampa ◽

Jared D. Acoba ◽

Dexi Chen ◽

Kelly Beemer ◽

Joel Gay ◽

...

Keyword(s):

Tumor Suppressor ◽

Mouse Model ◽

B Cell Lymphoma ◽

Tumor Formation ◽

Rank Test ◽

Serum Starvation ◽

Free Survival ◽

Induced Apoptosis ◽

Tumor Types

Abstract ASPP2 interacts with the tumor suppressor protein p53 and promotes damage-induced apoptosis in part through stimulation of p53-mediated apoptosis. We have previously demonstrated that low ASPP2 levels correlate with poor clinical outcome in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy. Moreover, reduced ASPP2 expression has been demonstrated in other tumor types. These findings led us to hypothesize that ASPP2 may function as a tumor suppressor. To further explore this, we targeted the ASPP2 allele in a mouse by homologous recombination using a knockout vector that replaced exons 10–17 with a neoR gene. Two separate ES clones were used for blastocyst injections to generate several chimeras that were used to generate ASPP2 heterozygous mice. ASPP2+/− mice appear developmentally normal and reproduce. However ASPP2−/− mice could not be generated. Genotype analysis as early as Ed 6.5 did not detect ASPP2−/− embryos---which implies an early embryonic lethal defect in the homozygote. ASPP2+/− (n=135) and ASPP2+/+ (n=63) sibling mice were observed for spontaneous tumor formation. Overall median tumor-free survival was 117 weeks in the ASPP2+/− mice verses 125 weeks in the ASPP2+/+mice (p = 0.035 log-rank test). Overall tumor incidence (at 115 weeks) for ASPP2+/− and ASPP2+/+ mice was 43% and 22%, respectively. The incidence of tumor types, from all tumors detected, was similar between ASPP2+/− and ASPP2+/+ mice: 34% versus 33% (lymphoma), 18% versus 14% (sarcoma), and 47% versus 52% (carcinoma), respectively. Compound p53+/−;ASPP2+/− mice did not exhibit accelerated tumor formation relative to p53+/−;ASPP2+/+ mice. Additionally, a tet-Myc:ASPP2+/− lymphoma mouse model did not exhibit accelerated lymphomagenesis. However, preliminary data suggests that ASPP2+/− mice may have an increased incidence of irradiation-induced leukemia/lymphoma when compared to ASPP2+/+ mice, and confirmatory studies are ongoing. In response to ionizing radiation, doxorubicin, or serum-starvation, preliminary analysis reveals a G0/G1 checkpoint defect in ASPP2+/− MEFs compared to ASPP2+/+ MEFs. Our results provide in vivo evidence that ASPP2 can function as a tumor suppressor. Further studies are underway to determine the mechanism of this observation.

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Filling the Gap Between Risk Assessment and Molecular Determinants of Tumor Onset

Carcinogenesis ◽

10.1093/carcin/bgaa135 ◽

2020 ◽

Author(s):

Federica Chiara ◽

Stefano Indraccolo ◽

Andrea Trevisan

Keyword(s):

Risk Assessment ◽

Cancer Biology ◽

Neoplastic Transformation ◽

Tumor Formation ◽

Cancer Risks ◽

The Past ◽

Tumor Onset ◽

Complex Process ◽

Molecular Determinants ◽

Carcinogen Exposure

Abstract In the past two decades, a ponderous epidemiological literature has causally linked tumor onset to environmental exposure to carcinogens. As consequence, risk assessment studies have been carried out with the aim to identify both predictive models of estimating cancer risks within exposed populations and establishing rules for minimizing hazard when handling carcinogenic compounds. The central assumption of these works is that neoplastic transformation is directly related to the mutational burden of the cell without providing further mechanistic clues to explain increased cancer onset after carcinogen exposure. Nevertheless, in the last few years, a growing number of studies have implemented the traditional models of cancer aetiology, proposing that neoplastic transformation is a complex process in which several parameters and crosstalk between tumor and microenvironmental cells must be taken into account and integrated with mutagenesis. In this conceptual framework, the current strategies of risk assessment that are solely based on the “mutator model” require an urgent update and revision to keep pace with advances in our understanding of cancer biology. We will approach this topic revising the most recent theories on the biological mechanisms involved in tumor formation in order to envision a roadmap leading to a future regulatory framework for a new, protective policy of risk assessment.

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Replication Study: Transcriptional amplification in tumor cells with elevated c-Myc

eLife ◽

10.7554/elife.30274 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 16

Author(s):

L Michelle Lewis ◽

Meredith C Edwards ◽

Zachary R Meyers ◽

C Conover Talbot ◽

Haiping Hao ◽

...

Keyword(s):

Gene Expression ◽

Tumor Cells ◽

Cancer Biology ◽

Statistical Significance ◽

Digital Gene Expression ◽

Original Study ◽

Silent Genes ◽

Before And After ◽

Meta Analyses ◽

Transcriptional Amplification

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Blum et al., 2015), that described how we intended to replicate selected experiments from the paper ‘Transcriptional amplification in tumor cells with elevated c-Myc’ (Lin et al., 2012). Here we report the results. We found overexpression of c-Myc increased total levels of RNA in P493-6 Burkitt’s lymphoma cells; however, while the effect was in the same direction as the original study (Figure 3E; Lin et al., 2012), statistical significance and the size of the effect varied between the original study and the two different lots of serum tested in this replication. Digital gene expression analysis for a set of genes was also performed on P493-6 cells before and after c-Myc overexpression. Transcripts from genes that were active before c-Myc induction increased in expression following c-Myc overexpression, similar to the original study (Figure 3F; Lin et al., 2012). Transcripts from genes that were silent before c-Myc induction also increased in expression following c-Myc overexpression, while the original study concluded elevated c-Myc had no effect on silent genes (Figure 3F; Lin et al., 2012). Treating the data as paired, we found a statistically significant increase in gene expression for both active and silent genes upon c-Myc induction, with the change in gene expression greater for active genes compared to silent genes. Finally, we report meta-analyses for each result.

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