Direct regulation of transforming growth factor β‐induced epithelial–mesenchymal transition by the protein phosphatase activity of unphosphorylated PTEN in lung cancer cells
Authors: Motohiro Kimura
Yoshinori Hasegawa
Tsutomu Kawabe
Norihito Omote
Kazuyoshi Imaizumi
Shinichi Miyazaki
Masaaki Kusunose
Koji Sakamoto
Ryo Ogata
Akira Ando
Naozumi Hashimoto
Daisuke Aoyama
Journal: Cancer Science (2015)
Discipline: CLINICAL MEDICINE
Keywords: PTEN; epithelial-mesenchymal transition; protein phosphatase activity; transforming growth factor β; β-Catenin.
Affiliations: Department of Respiratory Medicine Nagoya University Graduate School of Medicine Nagoya Japan, Department of Respiratory Medicine and Allergy Fujita Health University Toyoake Japan, Department of Pathophysiological Laboratory Sciences Nagoya University Graduate School of Medicine Nagoya Japan

Abstract:  Transforming growth factor β (TGFβ) causes the acquisition of epithelial-mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFβ, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFβ-induced phosphorylation of its C-terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C-terminus (PTEN4A), but not PTEN wild, inhibits TGFβ-induced EMT. Nevertheless, whether or not the blockade of TGFβ-induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C-terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFβ-induced EMT in lung cancer cells. The unphosphorylated PTEN C-terminus might not directly retain the phosphatase activities and repress TGFβ-induced EMT; the modification that keeps the PTEN C-terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFβ-induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C-terminus might be a therapeutic target to negatively regulate TGFβ-induced EMT in lung cancer cells. 

Retracted: No

PubPeer comments:
 **Primary Funding Source:** Private donors. **Role of the Funding Source** The funders did not contribute to design, collection, management, analysis, interpretation of data, writing of the report, or the decision to submit the report for publication. *** Could the authors perhaps share a few more details about the funders? The point of including the funding source is to be transparent about any potential influence, intended or otherwise, that may affect the independence of the research, leaving it to readers to make that judgement, not the authors. Example questions: Were the donors *individuals* previously unknown to the researchers? Do the individual donors have any economic, political or social ties that might create an impression of a conflict of interest? Are these *institutional* donors? If yes to the latter, what is the justification for not revealing the institution? Was the identity of the donor revealed to the editors or reviewers? Related to this series of questions, can the authors reveal if there a legal agreement that prevents disclosure of the donoru2019s name, and would that agreement be available, in redacted or non-redacted form, upon filing of a public records request? If yes, which author institution is a party to this agreement? , 
 #1 Actually I was able to find disclosure documents at the journal site (thanks!) from the authors which indirectly answered some of my questions. From author Boulware: ![file] *** Note the name Steve Kirsch. This is exactly my point about revealing funding sources. Mr. Kirsch is kindly contributing money toward searches for treatments for COVID-19, which I applaud. However, he has also been vocal about the possible utility of hydroxychloroquine, even penning an article on Medium. ![file] Link: https: medium.com @stkirsch why-drug-repurposing-is-our-best-hope-of-avoiding-a-mass-catastrophe-bad16f7e1945 Again, my point is not that Mr. Kirsch or any of the other funders necessarily influenced the results of the research, and in fact they may be disappointed in the results, but it seems to me that the terse u201cprivate fundersu201d disclosure in the manuscript leaves readers in the dark. , 
 We read the article intitled u201cHydroxychloroquine in nonhospitalized adults with COVID-19u201d, by Skipper et al, with much interest. We disagree with some of the authorsu2019 conclusions, which we believe could not be taken from the data shown. (1)tIn suppl table 3, concerning medication adherence, the sum of all categories in HCQ group is: 100% of the tablets, 157; 75-99% of the tablets, 8; 75% of the tablets, 16; never started medication, 22, so = 157+8+16+22= 203. In placebo group, the sum of these categories is 166+3+12+13=194. In suppl table 2, concerning the analysis of symptoms severity according to medication adherence, table states that in HCQ group, 165 patients took 75% of the tablets (15-19) and 38 75% (1-14), 165+38= 203. In placebo group, 169+25= 194. Comparing these two tables, it becomes clear that in suppl table 2 if category 75% was in fact 1-14 tablets, we should be missing 22 patients in group HCQ and 13 patients in placebo group, who never started medication (0 tablets). Since we are not missing them, in both tables group HCQ has 203 patients and group placebo has 194 patients, I assume the author kept patients who never started medication in HCQ and placebo groups. It is not a question of writing 0-14 instead of 1-14 tablets; it is about excluding patients who did not start treatment from ITT analysis. The u201c1-14 tabletsu201d induces the reader to assume that authors performed the usual analysis, excluding patients who did not start treatment. We recall that even in intention-to-treat analysis, if no treatment was applied at all, the usual procedure is to exclude these patients from the full analysis set. https: www.clinfo.eu itt-vs-pp (2)tWe acknowledge that ignoring those patients who stopped the medicine because of side effects introduces biases. However, it does not apply to this situation, since these 22 patients who never started HCQ treatment never experienced any side effects and there were also 13 patients who never started placebo; they did not start treatment for reasons not related to group allocation. While no biases might arise from excluding patients who never started treatment allocated to both HCQ and placebo groups, keeping them in the full analysis set might mask any positive HCQ effects. Masking the positive effects of a drug during a pandemic is a harmful choice. (3)tIf a medication is found to work under optimal conditions in the trial setting, the adherence to this medication in the field will be far better. We recall that the uncertainty about the efficacy of a drug being tested in the trial setting may mean that trial participants are less likely to persevere with unpleasant side effects of treatment than those who have been assured of the efficacy of their treatment (1). Yet the authors seem to disregard their own HCQ positive results found when patients taking 75% of the tablets are considered, a significant 19.5% relative benefit in symptom severity (two-tailed p-value=0.022). (4)tAuthors u201ccaution against over-interpretation of this resultu201d, a 19.5% relative benefit in symptom severity, arguing that u201cthis [adherent HCQ] group did not improve any more than the non-adherent hydroxychloroquine group nor the non-adherent placebo groupu201d. However, unlike comparison between treated and untreated group, the comparison between adherent and non-adherent group is invalid as the latter groups are not selected by randomization and are subject to u201cnon-adherence biasu201d, i.e., non-adherent patients might have not taken the drug for instance because they were healthier than adherent patients. This is corroborated by the data in the table which shows that the small non-adherent group did overall much better than the larger adherent group. (5)tWe understand that the proper way to analyze the data is by doing both an analysis of the intention-to-treat population (excluding, as usual, patients who took zero tablets) and a per protocol analysis of the patients with 75% adherence to the treatment. The consequence of these considerations is that conclusions such as the one in the abstract and discussion, u201chydroxychloroquine did not substantially reduce symptom severity in outpatients with early, mild COVID-19u201d, cannot be assumed when a possible positive effect might arise from the rightful exclusion of patients who never started treatment and there is a clear 19.5% effect of hydroxychloroquine over placebo when patients who took the tablets are compared, i.e., per-protocol analysis. 1.tDodd S, White IR, Williamson P. Nonadherence to treatment protocol in published randomised controlled trials: a review. Trials. 2012;13:84. Claudia Neto Paiva u2013 Associate Professor, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Brazil Daniel Victor Tausk u2013 Associate Professor, Instituto de Matemu00e1tica e Estatu00edstica, Universidade de Su00e3o Paulo , 
 Correction In the article by Skipper and colleagues (1), there was a typo in Supplement Table 2 regarding medication adherence. One of the labels in the first column was listed as u201c 75% (1-14 tablets)u201d and has been corrected to u201c 75% (0-14 tablets).u201d This is still 75% adherence, and the breakdown is displayed in Supplement Table 3. These corrections were made to the online version on 8 October 2020. , 
 #2, Actinopolyspora, there is a clearly disclosed financial support section in the print (PDF) version of the manuscript. I would agree in the web page version, this is not apparent. , 
 I read the comments [1] on the manuscript and I see that Skipper et al. accuse Dr. Wiseman of altering the data and manipulation in a post-hoc analysis [2] of the companion post-exposure trial [3]. On twitter [4], one of the authors promoted a  mITT reanalysis of their own data in which it appears that lost to follow-up were assumed not have the outcome in the placebo group (as in the ITT analysis presented in the paper), while they were removed from the HCQ group. This not a valid mITT analysis and this is quite misleading. in [5], the time of exposure was conveniently redefined as *last* exposure. I am not sure whether this is less misleading than the reanalysis of Wiseman et al. In the present manuscript, Skipper et al. compare the change in symptoms severity to that of oseltamivir in Influenza. However, the paper cited was about oseltamivir given within 48 hours after symptom onset (it is know to be ineffective beyond that narrow window). For a proper comparison, it would be important to look at patients in Skipper et al. who started the treatment within two days, but lack of information on shippment delays makes it impossible. It is reasonable to guess that the patients who could start treatment the earliest after symptom onset are those who were asymptomatic when they enrolled (for the companion trial), but who developed symptoms during the shipping of the drugs. In the supplemental material, it appears it is that group of patients who had the largest benefit indeed. [1] https: www.acpjournals.org doi full 10.7326 M20-4207#_comments [2] Wiseman DM, et al., Effective post-exposure prophylaxis of Covid-19 is associated with use of hydroxychloroquine: Prospective re-analysis of a public dataset incorporating novel data. medRxiv 2020 http: doi.org 10.1101 2020.11.29.20235218 [3] Boulware DR, et al. A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19. N Engl J Med 2020; 383:517-25. http: doi.org 10.1056 NEJMoa2016638 [4] https: twitter.com boulware_dr status 1311332673969848325 [5] Nicol MR, Boulware DR, Rajasingham R. Reply to author. Clin Infect Dis. 2020; In Press. doi:10.1093 cid ciaa1809. ,