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

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: Melanoma genome sequencing reveals frequent PREX2 mutations

eLife ◽

10.7554/elife.21634 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 19

Author(s):

Stephen K Horrigan ◽

Pascal Courville ◽

Darryl Sampey ◽

Faren Zhou ◽

Steve Cai ◽

...

Keyword(s):

Genome Sequencing ◽

Cancer Biology ◽

Tumor Formation ◽

Original Study ◽

Free Survival ◽

Tumor Onset ◽

Study Evaluation ◽

Significant Difference ◽

Meta Analyses

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: 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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Replication Study: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology

eLife ◽

10.7554/elife.51019 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

John Kerwin ◽

Israr Khan ◽

Elizabeth Iorns ◽

Rachel Tsui ◽

Alexandria Denis ◽

...

Keyword(s):

Cancer Biology ◽

Cancer Cell Line ◽

Prostate Cancer Cell Line ◽

Original Study ◽

Mrna Levels ◽

Independent Function ◽

Tumour Biology ◽

Pten Mrna ◽

Du145 Cells ◽

Meta Analyses

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Khan et al., 2015), that described how we intended to replicate selected experiments from the paper "A coding-independent function of gene and pseudogene mRNAs regulates tumour biology" (Poliseno et al., 2010). Here we report the results. We found PTEN depletion in the prostate cancer cell line DU145 did not detectably impact expression of the corresponding pseudogene PTENP1. Similarly, depletion of PTENP1 did not impact PTEN mRNA levels. The original study reported PTEN or PTENP1 depletion statistically reduced the corresponding pseudogene or gene (Figure 2G; Poliseno et al., 2010). PTEN and/or PTENP1 depletion in DU145 cells decreased PTEN protein expression, which was similar to the original study (Figure 2H; Poliseno et al., 2010). Further, depletion of PTEN and/or PTENP1 increased DU145 proliferation compared to non-targeting siRNA, which was in the same direction as the original study (Figure 2F; Poliseno et al., 2010), but not statistically significant. We found PTEN 3'UTR overexpression in DU145 cells did not impact PTENP1 expression, while the original study reported PTEN 3'UTR increased PTENP1 levels (Figure 4A; Poliseno et al., 2010). Overexpression of PTEN 3'UTR also statistically decreased DU145 proliferation compared to controls, which was similar to the findings reported in the original study (Figure 4A; Poliseno et al., 2010). Differences between the original study and this replication attempt, such as level of knockdown efficiency and cellular confluence, are factors that might have influenced the results. Finally, where possible, 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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Registered report: Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs

eLife ◽

10.7554/elife.06959 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 2

Author(s):

Irawati Kandela ◽

James Chou ◽

Kartoa Chow ◽

Keyword(s):

Cell Death ◽

Body Weight ◽

Cancer Biology ◽

Scientific Research ◽

Open Science ◽

Cancer Drugs ◽

Tumor Weight

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered report describes the proposed replication plan of key experiments from ‘Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs’ by Sugahara and colleagues, published in Science in 2010 (<xref ref-type="bibr" rid="bib10">Sugahara et al., 2010</xref>). The key experiments being replicated include Figure 2 and Supplemental Figure 9A. In Figure 2, Sugahara and colleagues presented data on the tumor penetrance of doxorubicin (DOX) when co-administered with the peptide iRGD, as well as the effect of co-treatment of DOX and iRGD on tumor weight and cell death. In Supplemental Figure 9A, they tracked body weight of mice treated with DOX and iRGD to provide evidence that iRGD does not increase known DOX toxicity. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

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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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Replication Study: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs

eLife ◽

10.7554/elife.56651 ◽

2020 ◽

Vol 9 ◽

Author(s):

Hongyan Wang ◽

Hanna S Radomska ◽

Mitch A Phelps ◽

Elizabeth Iorns ◽

Rachel Tsui ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Suppressor ◽

Cancer Biology ◽

Hct116 Cell ◽

Original Study ◽

Wild Type ◽

Pten Protein ◽

Protein Levels ◽

Meta Analyses ◽

Hct116 Cells

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Phelps et al., 2016) that described how we intended to replicate selected experiments from the paper ‘Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs’ (Tay et al., 2011). Here, we report the results. We found depletion of putative PTEN competing endogenous mRNAs (ceRNAs) in DU145 cells did not impact PTEN 3’UTR regulation using a reporter, while the original study reported decreased activity when SERINC1, VAPA, and CNOT6L were depleted (Figure 3C; Tay et al., 2011). Using the same reporter, we found decreased activity when ceRNA 3’UTRs were overexpressed, while the original study reported increased activity (Figure 3D; Tay et al., 2011). In HCT116 cells, ceRNA depletion resulted in decreased PTEN protein levels, a result similar to the findings reported in the original study (Figure 3G,H; Tay et al., 2011); however, while the original study reported an attenuated ceRNA effect in microRNA deficient (DicerEx5) HCT116 cells, we observed increased PTEN protein levels. Further, we found depletion of the ceRNAs VAPA or CNOT6L did not statistically impact DU145, wild-type HCT116, or DicerEx5 HCT116 cell proliferation. The original study reported increased DU145 and wild-type HCT116 cell proliferation when these ceRNAs were depleted, which was attenuated in the DicerEx5 HCT116 cells (Figure 5B; Tay et al., 2011). Differences between the original study and this replication attempt, such as variance between biological repeats, are factors that might have influenced the results. Finally, we report meta-analyses for each result.

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Replication Study: BET bromodomain inhibition as a therapeutic strategy to target c-Myc

eLife ◽

10.7554/elife.21253 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 23

Author(s):

Fraser Aird ◽

Irawati Kandela ◽

Christine Mantis ◽

Keyword(s):

Overall Survival ◽

Cancer Biology ◽

Therapeutic Strategy ◽

Early Death ◽

Vehicle Control ◽

Tumor Burden ◽

Original Study ◽

Control Group ◽

Minimal Impact ◽

Significant Difference

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 "BET bromodomain inhibition as a therapeutic strategy to target c-Myc" (Delmore et al., 2011). Here we report the results of those experiments. We found that treatment of human multiple myeloma (MM) cells with the small-molecular inhibitor of BET bromodomains, (+)-JQ1, selectively downregulated MYC transcription, which is similar to what was reported in the original study (Figure 3B; Delmore et al., 2011). Efficacy of (+)-JQ1 was evaluated in an orthotopically xenografted model of MM. Overall survival was increased in (+)-JQ1 treated mice compared to vehicle control, similar to the original study (Figure 7E; Delmore et al., 2011). Tumor burden, as determined by bioluminescence, was decreased in (+)-JQ1 treated mice compared to vehicle control; however, while the effect was in the same direction as the original study (Figure 7C-D; Delmore et al., 2011), it was not statistically significant. The opportunity to detect a statistically significant difference was limited though, due to the higher rate of early death in the control group, and increased overall survival in (+)-JQ1 treated mice before the pre-specified tumor burden analysis endpoint. Additionally, we evaluated the (−)-JQ1 enantiomer that is structurally incapable of inhibiting BET bromodomains, which resulted in a minimal impact on MYC transcription, but did not result in a statistically significant difference in tumor burden or survival distributions compared to treatment with (+)-JQ1. Finally, we report meta-analyses for each result.

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Replication Study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate

eLife ◽

10.7554/elife.26030 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 9

Author(s):

Megan Reed Showalter ◽

Jason Hatakeyama ◽

Tomas Cajka ◽

Kacey VanderVorst ◽

Kermit L Carraway ◽

...

Keyword(s):

Enzyme Activity ◽

Vector Control ◽

Cancer Biology ◽

Original Study ◽

Idh Mutation ◽

Wild Type ◽

The Common ◽

Idh1 And Idh2 Mutations ◽

Mutant Idh1 ◽

Meta Analyses

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (<xref ref-type="bibr" rid="bib14">Fiehn et al., 2016</xref>), that described how we intended to replicate selected experiments from the paper "The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate" (Ward et al., 2010). Here, we report the results of those experiments. We found that cells expressing R172K mutant IDH2 did not display isocitrate-dependent NADPH production above vector control levels, in contrast to the increased production observed with wild-type IDH2. Conversely, expression of R172K mutant IDH2 resulted in increased alpha-ketoglutarate-dependent consumption of NADPH compared to wild-type IDH2 or vector control. These results are similar to those reported in the original study (Figure 2; Ward et al., 2010). Further, expression of R172K mutant IDH2 resulted in increased 2HG levels within cells compared to the background levels observed in wild-type IDH2 and vector control, similar to the original study (Figure 3D; Ward et al., 2010). In primary human AML samples, the 2HG levels observed in samples with mutant IDH1 or IDH2 status were higher than those observed in samples without an IDH mutation, similar to what was observed in the original study (Figure 5C; Ward et al., 2010). Finally, we report meta-analyses for each result.

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