The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44
Authors: Dean G Tang
Hong Yan
Andreas G Bader
Collene Jeter
Randy Fagin
David Brown
Xin Chen
Sofia Honorio
Tammy Calhoun-Davis
Bigang Liu
Can Liu
Kevin Kelnar
Hangwen Li
Jason F Wiggins
Lubna Patrawala
Journal: Nature medicine (2011)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Cancer stem cells; MicroRNAs; CD44; Prostate cancer; Metastasis
Affiliations: Department of Molecular Carcinogenesis, the University of Texas M.D. Anderson Cancer Center, Science Park, Smithville, Texas, USA.

Abstract:  Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs. 

Retracted: No

PubPeer comments:
 #1 MAY 23, 2019 SUBJECT: Response to u201cReplication Study: The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44 (Yan et al., eLIFE, 2019;8:e43511)u201d FROM: Dean G. Tang, M.D, Ph.D; Can Liu, Ph.D Recently our paper u201cThe microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44u201d, published in 2011 in Nature Medicine (17:211-215), was evaluated for replication by the Reproducibility Project and published in eLife by Yan et al (see above). In our original paper, we did an unbiased miRNA profiling for 324 miRNAs in cancer stem cell enriched populations using prostate cancer xenograft cells including LAPC9, LAPC4, and Du145. We found that miR-34a was one of the most under-expressed miRNAs in CD44 or CD133 enriched cancer stem cell populations. To further validate the under-expression and to determine its clinical relevance, we collected human primary prostate cancer cells freshly purified out from 20 patient tumors, and compared miR-34a expression in CD44-positive and CD44-negative cells. The results were consistent with findings in xenograft models, demonstrating that miR-34a is indeed under-expressed in CD44 positive cells. Each experiment in our original paper was rigorously designed and performed with biological and technical repeats (see Summary in Supplementary Fig. 1), adding robustness to our analysis. Overall, we stand strongly behind our original studies. A few years ago, the Reproducibility Project selected our paper to test whether the results would be reproducible. We were very proud of our work and supportive of these efforts. We contributed to these efforts by providing some of the key reagents and by sharing experimental protocols and insights. The recently published results in eLIFE, however, are disappointing. Not only did the authors not design the right experiments to truly reproduce our findings, but they deviated significantly from our original work, and the work they presented was not done with same rigorous standards set forth in our original paper. Therefore, for the reasons outlined below we have serious concerns about the ability of the Reproducibility Project to handle the scope of their stated mission, and hereby contest the recently reported results in the Replication Study. In the published Replication Study, the authors used only one model system to u2018verifyu2019 the under-expression of miR-34a in prostate cancer cells, specifically, the LAPC4 model. In contrast, our original paper utilized numerous prostate cancer cell lines or xenograft models including LAPC4, LAPC9, DU145, as well as freshly purified patient prostate cancer cells. This issue has been raised by us and other reviewers in the original review process for the registered report protocol published in 2015. Since it would be difficult to include patient derived tumor cells, we suggested that adding at least one more cell line model, such as Du145 (the main model of our original study), would be feasible and should be done. However, this did not happen because u201cDue to constraints of budget and time, we decided to restrict our xenograft experiments to only the LAPC4 cell lines.u201d (From email communication with Fraser Tan, the project coordinator from the replication group at that time). Most importantly, as pointed out by all the reviewers for this Replication Study, the research group responsible for replicating our work switched the source of LAPC4 cells, from what we originally provided to that of another LAPC4 variant from a different lab. Our LAPC4 cells have been validated by cell line authentication at our institute (MD Anderson Cancer Center, and Roswell Park Comprehensive Cancer Center), and have been intensively used in our lab for more than 12 years. We have no problem generating xenograft tumors with these cells. The author s response to reviewers that u201cWe attempted to reach out to the original authors again, but were informed that work on miR-34a and LAPC4 cells had stopped in the Tang lab and, unfortunately, they could not assist us.u201d is not true. We did not receive an email or communications from the authors about this issue. We, as a research lab focusing on prostate cancer, have never stopped using LAPC4 as one of our xenograft models. If we had been contacted, we would have been happy to help them figure out any potential problems and provided another vial of cells or xenograft tumor pieces for use in the replication study. We acknowledge there seems to be some drift in the STR profiling of our cells as we have seen this as well, but we have also compared multiple sources and find this subtle variation to be present in all the LAPC4 sources we have profiled. More importantly, the profiling of our LAPC4 line is consistent when collected over time, suggesting little genetic drift. Cell line variations in research are not uncommon, and it is important to the integrity of the original studies to use the original source cells. Altering the source of the LAPC4 model is a serious concern associated with this Replication Study, and in our opinion not adequately addressed. One could argue that this can be the grounds for dismissal of the entire reproducibility efforts. As to why the originally supplied vial of LAPC4 cells failed to generate tumors, there may be many reasons. It is possible that after long distance transportation from the States to China (where the u2018Reproducibilityu2019 studies were presumably conducted), the cryopreserved cells might have been stressed and lost viability. Shipment induced stress may require longer times in the NOD SCID mice to fully recover. From our previous experience, xenograft tumor take rate from long-term stored frozen vials is drastically reduced compared to freshly purified cells. Itu2019s possible that the lot of cryopreserved cells was bad. Regardless, we have this model, continue to use this model, and would have been happy to supply another vial upon request. Notably, we rarely use in vitro cultured LAPC4 cells for experiments. Most of the time, we use freshly purified cells from xenograft tumors, limiting in vitro expansion. In addition to altering the source of the original cell model for these replication studies, there are additional reasons for concern relating to this Replication Project. For several experiments published in this Replication Study, the authors failed to include proper biological replicates for their experiments. For example, in Figure 1, which tested miR-34a expression in CD44 populations, both panels were generated based on a one-time N=1 experiment. The authors use this N=1 experiment to conclude that our original findings from multiple models, with multiple biological and technical replicates (Supplementary Fig. 1), are not reproducible. This is unacceptable. There are very few, if any, reputable journals that would publish an original study with an N=1 experiment, so why is this acceptable when it comes to a replication study? These results hurt the credibility of our original study, and it is, in our opinion, callous to pawn this off as a reproducibility study. We believe all published work deserves at least a minimum of 3 attempted replicates before being discounted. Again, to perform N=1 is sub-standard, and we are concerned that this did not receive more attention in the review process of this replication study. In addition to the N=1 sample size, the authors again took it upon themselves to change the parameters of the original study. Our original study gated the top 10% of CD44-positive cells and compared against the bottom 10% of CD44-positive cells, or in this case CD44-negative. The authors here compared the top 1% of positive cells with the bottom 99% of cells, or everything else. This is again a drastic shift from the originally published work. Given the authoru2019s use of alternatively sourced LAPC4 cells, and the changes to original experimental parameters, these studies cannot, and should not, be considered as an attempt to validate replicate our original findings. Continuing to build on their findings, the authors next attempted to replicate our findings that miR-34a overexpression inhibited tumorigenicity of the LAPC4 model. Authors validated over expression of miR-34a prior to injecting cells into mice, but found no difference in tumorigenicity with miR-34a overexpression. Authors proceeded to state that this demonstrates that original results were unable to be replicated. This is clearly not true since they subsequently show that the miR-34a expressing tumor group actually lacked miR-34a overexpression. In fact, they show loss of miR-34a expression, suggesting there is an active attempt to suppress this expression by tumors. To be fair, we have to point out that we also observed the loss of miR-34a expression in endpoint tumors. This data was part of the discussions presented in Dr. Can Liuu2019s PhD dissertation. Consistent with the suggestions made by reviewers of this Replication study, the loss of over-expression of miR-34a does not conflict with the tumor suppressive functions of miR-34a. On the contrary, miR-34a silencing strongly suggests that over-expression represents a negative factor against tumor growth and progression, which is consistent with the main findings of our original paper that miR-34a suppresses tumor development. All 4 reviewers of this replication study pointed out some of these major concerns. As noted by reviewer #2, u201cThis experimental observation does not allow one to draw the conclusion that miR-34 does not suppress tumor growth or progression in this system, since miR-34 over-expression is not confirmed in vivo.u201d Unfortunately, the reviewers and editors did not find that these flaws were sufficient for the Replication study to not be published. We find it puzzling that the scientific rigor and reproducibility standards seemed to be much lower for this publication than they were for the original work, and opposed it being published for the reasons outlined above. Needless to say, the tumor suppressive functions of miR-34a have been intensively reported and corroborated in many systems by independent researchers. Since the publication of the original paper, there have been 30 publications that are directly or indirectly in support of the original findings regarding miR-34a targeting CD44 (an incomplete list is included below). All of these studies in essence have reproduced our original findings, which further contradicts and invalidates the findings associated with the current Replication study in eLIFE. We believe that everything we published in our original paper should be reproducible and we stand strongly behind the tumor suppressive functions of miR-34a in prostate cancer (stem) cells. Our original paper presented extensive experimental evidence to prove that over-expression of miR-34a suppressed tumor development, in multiple model systems, not just one (Supplementary Fig. 1). We performed u201cgain of functionu201d assays by over-expressing miR-34a using either miRNA mimics or lenti-viral and retroviral mediated induction in multiple prostate cancers models, including LAPC9, LAPC4, DU145, PPC-1, PC3 and even a primary patient derived tumor xenograft cell, HPCa58. Furthermore, a more drastic tumor inhibitory effect was observed when we over-expressed miR-34a in CD44+ DU145 and CD44+ LAPC9 cells, possibly better suited models to demonstrate reproducibility of the original findings. We applaud conceptually the Reproducibility Project initiative and we did work extensively with the group in the early phase of this replication project. However, the replication team must take a higher level of responsibility for replication experiments. Experiments must be designed and conducted with ultimate rigor to guarantee their own reproducibility. Otherwise, misleading and or wrong conclusions will be drawn, and the scientific field will be muddied and misguided. Failure to maintain high standards is unfortunate and defeats the intended purpose of the Reproducibility Project. Due to the major issues identified above, we find that this Reproducibility Project study is highly flawed, and contrary to the original intensions does very little to help the scientific community or greater public good. u2022tmicroRNA-34a suppresses invasion and metastatic in esophageal squamous cell carcinoma by regulation CD44. Zuo J, Zhu K, Wang Y, Yu Z. Mol Cell Biochem. 2018 Jun;443(1-2):139-149. doi: 10.1007 s11010-017-3218-3. Epub 2017 Nov 1. u2022tMicroRNA and transcriptome analysis in periocular Sebaceous Gland Carcinoma. Bladen JC, Wang J, Sangaralingam A, Moosajee M, Fitchett C, Chelala C, Beaconsfield M, O Toole EA, Philpott MP, Ezra DG. Sci Rep. 2018 May 14;8(1):7531. doi: 10.1038 s41598-018-25900-z. u2022tNanovesicle-mediated systemic delivery of microRNA-34a for CD44 overexpressing gastric cancer stem cell therapy. Jang E, Kim E, Son HY, Lim EK, Lee H, Choi Y, Park K, Han S, Suh JS, Huh YM, Haam S. Biomaterials. 2016 Oct;105:12-24. doi: 10.1016 j.biomaterials.2016.07.036. Epub 2016 Jul 30. u2022tmiR-34a inhibits differentiation of human adipose tissue-derived stem cells by regulating cell cycle and senescence induction. Park H, Park H, Pak HJ, Yang DY, Kim YH, Choi WJ, Park SJ, Cho JA, Lee KW. Differentiation. 2015 Nov-Dec;90(4-5):91-100. doi: 10.1016 j.diff.2015.10.010. Epub 2015 Dec 8. u2022tDepletion of histone deacetylase 1 inhibits metastatic abilities of gastric cancer cells by regulating the miR-34a CD44 pathway. Lin L, Jiang H, Huang M, Hou X, Sun X, Jiang X, Dong X, Sun X, Zhou B, Qiao H. Oncol Rep. 2015 Aug;34(2):663-72. doi: 10.3892 or.2015.4010. Epub 2015 May 28. u2022tMicroRNA-34a functions as an anti-metastatic microRNA and suppresses angiogenesis in bladder cancer by directly targeting CD44. Yu G, Yao W, Xiao W, Li H, Xu H, Lang B. J Exp Clin Cancer Res. 2014 Dec 31;33:779. doi: 10.1186 s13046-014-0115-4. u2022tmiRNA-34a suppresses cell proliferation and metastasis by targeting CD44 in human renal carcinoma cells. Yu G, Li H, Wang J, Gumireddy K, Li A, Yao W, Tang K, Xiao W, Hu J, Xiao H, Lang B, Ye Z, Huang Q, Xu H. J Urol. 2014 Oct;192(4):1229-37. doi: 10.1016 j.juro.2014.05.094. Epub 2014 May 24. u2022tCisplatin-induced epigenetic activation of miR-34a sensitizes bladder cancer cells to chemotherapy. Li H, Yu G, Shi R, Lang B, Chen X, Xia D, Xiao H, Guo X, Guan W, Ye Z, Xiao W, Xu H. Mol Cancer. 2014 Jan 15;13:8. doi: 10.1186 1476-4598-13-8. Erratum in: Mol Cancer. 2014;13():183. u2022tThe CD44(high) tumorigenic subsets in lung cancer biospecimens are enriched for low miR-34aexpression.Basak SK, Veena MS, Oh S, Lai C, Vangala S, Elashoff D, Fishbein MC, Sharma S, Rao NP, Rao D, Phan R, Srivatsan ES, Batra RK. PLoS One. 2013 Sep 3;8(9):e73195. doi: 10.1371 journal.pone.0073195. eCollection 2013. Erratum in: PLoS One. 2013;8(9). u2022tRepression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness. Siemens H, Jackstadt R, Kaller M, Hermeking H. Oncotarget. 2013 Sep;4(9):1399-415. u2022tmiR-34a inhibits the metastasis of osteosarcoma cells by repressing the expression of CD44. Zhao H, Ma B, Wang Y, Han T, Zheng L, Sun C, Liu T, Zhang Y, Qiu X, Fan Q. Oncol Rep. 2013 Mar;29(3):1027-36. doi: 10.3892 or.2013.2234. Epub 2013 Jan 11. u2022tTargeted expression of miR-34a using the T-VISA system suppresses breast cancer cell growth and invasion. Li L, Xie X, Luo J, Liu M, Xi S, Guo J, Kong Y, Wu M, Gao J, Xie Z, Tang J, Wang X, Wei W, Yang M, Hung MC, Xie X. Mol Ther. 2012 Dec;20(12):2326-34. doi: 10.1038 mt.2012.201. Epub 2012 Oct 2. u2022tmiR-34 and SNAIL form a double-negative feedback loop to regulate epithelial-mesenchymal transitions. Siemens H, Jackstadt R, Hu00fcnten S, Kaller M, Menssen A, Gu00f6tz U, Hermeking H. Cell Cycle. 2011 Dec 15;10(24):4256-71. doi: 10.4161 cc.10.24.18552. Epub 2011 Dec 15. u2022tRestitution of tumor suppressor microRNAs using a systemic nanovector inhibits pancreatic cancer growth in mice. Pramanik D, Campbell NR, Karikari C, Chivukula R, Kent OA, Mendell JT, Maitra A. Mol Cancer Ther. 2011 Aug;10(8):1470-80. doi: 10.1158 1535-7163.MCT-11-0152. Epub 2011 May 27. Sincerely, Dean G. Tang, M.D., Ph.D Professor and Chairman Department of Pharmacology Therapeutics Roswell Park Comprehensive Cancer Center Buffalo, NY U.S.A , 
 Results from this paper could not be reproduced by the Reproducibility Project: Cancer Biology (10.7554 eLife.43511 https: elifesciences.org articles 43511 )  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 u2018The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44u2019 (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 3u2019UTR 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 3u2019UTR (Figure 4D; Liu et al., 2011). Finally, where possible, we report meta-analyses for each result. ,