Targeting of PYK2 Synergizes with EGFR Antagonists in Basal-like TNBC and Circumvents HER3-Associated Resistance via the NEDD4-NDRG1 Axis
Authors: Sungyoung Shin
Gordon B Mills
Anna-Katharina Müller
Effrosini Panayotopoulou
Charu Kothari
Nandini Verma
Sima Lev
Amir Kedan
Uwe Holtrich
Thomas Karn
Lan K Nguyen
Michael Selitrennik
Journal: Cancer research (2017)
Discipline: CLINICAL MEDICINE
Keywords: Triple-negative breast cancer; TNBC; EGFR; PYK2; FAK
Affiliations: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel., Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas., Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia., Department of Obstetrics and Gynecology, Goethe University Frankfurt, Frankfurt, Germany., Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. .

Abstract:  Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous disease with poor prognosis and no effective targeted therapies. EGFR is highly expressed in basal-like TNBC and is considered as a potential therapeutic target. However, EGFR targeting exerts only marginal clinical benefits, possibly due to activation of compensatory signaling pathways, which are frequently associated with HER3 upregulation. Here we show that concomitant targeting of EGFR and the nonreceptor tyrosine kinases PYK2/FAK synergistically inhibits the proliferation of basal-like TNBC cells in vitro and attenuates tumor growth in a mouse xenograft model. Dual targeting of EGFR and PYK2/FAK inhibited complementary key growth and survival pathways mediated by AKT, S6K, STAT3, and ERK1/2 activation. PYK2 inhibition also abrogated HER3 upregulation in response to EGFR antagonists, thereby circumventing HER3-associated drug resistance. Mechanistically, PYK2 inhibition facilitated the proteasomal degradation of HER3 while inducing upregulation of NDRG1 (N-myc downstream regulated 1 gene). NDRG1 enhanced the interaction of HER3 with the ubiquitin ligase NEDD4, while PYK2, which interacts with NEDD4 and HER3, interfered with NEDD4-HER3 binding, suggesting that the PYK2-NDRG1-NEDD4 circuit has a critical role in receptor degradation, drug response, and resistance mechanism. Our studies offer a preclinical proof of concept for a strategy of cotargeting the EGFR and PYK2/FAK kinases to improve TNBC therapy. Cancer Res; 77(1); 86-99. ©2016 AACR. 

Retracted: No

PubPeer comments:
 In this paper the authors show that MDAMB468 and BT20 breast cancer cell lines are not affected by Gefitinib treatment. During years Gefitinib has been used for cancer treatment and it has been shown as a powerful inhibitor of breast cancer cell lines growth. For example in Journal of Cancer 2014, Sohn shows that 1 uM of Gefintinib inhibits more than 90% of cell growth **[1]** In the figure 1- D authors show a staining with crystal violet to demonstrate the effect of Gefitinib. ![file] , 
 #1 Figure 1F:  Colony formation assay was used to demonstrate the stimulatory effect of PF4313196 on gefitinib-induced **MDA-MB-468** or **BT-20** cell death. Representative images of crystal violet staining of control and drug-treated cells (72 hours). ![file] Plates of (MDA-MB-468 + 1 micromol L of gefitinib) and (BT-20 + 10 micromol L) are more similar than one would expect. , 
 #2 I look at the paper and this article has almost 500 blots in the main figures and in supplementary material, which is itself a huge amount of data to produce and manage.....its no surprise and even obvious that few mistakes happened. After reading the article, I would rather congratulate the authors for such a great and comprehensive study, which actually deserves a much higher journal than Cancer research. Putting research articles on a forum like Pubpeer for such a stupid reason is too silly and non-scientific. It is unfortunate, that people are using the pubpeer for highlighting graphical mistakes rather than the scientific content and message which is overlooked. This is so funny to imagine....how does a total protein filliped gel picture matters to these findings. If the authors produced 500 blots for the article that means they might have 5000 blots in spare which can be used as STAT3 total protein IB for this particular piece of IB, so tell me why they would be sooo lazy to flip and reuse JUST the adjutant STAT3 in the same figure intentionally? Mr. Condylocarpon Amazonicum, either you have a plenty of time to waste on non important things or you are obsessed about STAT3 immunoblots of the same authors again and again in different articles. Very unfortunately, the purpose of such Pubpeer posts appears to me more oriented motive for personal revenge rather than the improvement of science. , 
 #5 Thank you for taking the time to bring your thoughts here. Perhaps not unsurprisingly, I disagree. *I look at the paper and this article has almost 500 blots in the main figures and in supplementary material, which is itself a huge amount of data to produce and manage.....its no surprise and even obvious that few mistakes happened.* The point is that it is not really clear how many blots there are. I havenu2019t looked at the supplement yet though. But some of these WB slices have visible but unacknowledged lane splicing, e.g. Inputs in Fig. 6G. Prior to 2004, practically everybody did this. Almost all publishers now explicitly disallow this manipulation because too often they got their fingers burnt, so to speak. *After reading the article, I would rather congratulate the authors for such a great and comprehensive study, which actually deserves a much higher journal than Cancer research.* We are not supposed to pay attention to impact factor nowadays. (Itu2019s 9.130 to a statistically absurd 3 decimal places.) But I accept that we should try to publish in good well-edited journals and so I will now bear in mind that **Cancer Research** is a poor and lowly place to be seen to publish in. *Mr. Condylocarpon Amazonicum, either you have a plenty of time to waste on non important things or you are obsessed about STAT3 immunoblots of the same authors again and again in different articles. Very unfortunately, the purpose of such Pubpeer posts appears to me more oriented motive for personal revenge rather than the improvement of science.* It is the Erk immunoblots that I have usually found to be most irksome. Many of these were removed from the literature when the nine Weizmann institute papers were retracted in one day https: retractionwatch.com 2017 05 25 journal-retracts-nine-papers-one-day-author-investigation-weizmann-institute And then there are the fabricated blots in the fourth worst-ever Weizmann paper (now retracted) which elicited a crazy and potentially very dangerous clinical trial attempting to induce an autoimmune reaction to a cellular heat shock factor https: pubpeer.com publications 08B08B9681EE15361E3BBC71938605 And therefore this is absolutely about working towards the improvement of how science gets published. It is only by showing how bad the current situation is that we can hope to get an improvement. People need to stop making stuff up. Electronic publishing means that there is now no bar to making the original data available. Original time-stamped, metadata that links to the researcher who did the experiment. But it needs scientists, research institutes, publishers and funders to move forward together. The amount of time that *C. amazonicum* has to waste on this unimportant stuff is, it regrets, quite finite. Therefore, like some others who regularly comment here, *C. amazonicum* prefers to go to where the low hanging fruit is. And there is surely plenty more to be harvested from these orchards. , 
 * people are using the pubpeer for highlighting graphical mistakes rather than the scientific content and message which is overlooked.  * If one cannot trust the graphical components, how can one trust the results that the authors obtained from them? * the purpose of such Pubpeer posts appears to me more oriented motive for personal revenge rather than the improvement of science * Revenge? For what? It is certainly **possible** that one of the paper s authors has done injury to Condylocarpon Amazonicum, an injury or insult which C. Amazonicum nurtured in his or heart over many years (on the  dish best served cold principle), until vengeance is finally obtained by criticizing Figures in a scientific publication. But we need more details before accepting that explanation. , 
 #3 Very good spots of the spots, Wax Plant and Javanese Bat. The paper is so recent that the original high resolution images, replete with time stamps and sundry other metadata will surely all be neatly stored in the groupu2019s LIMS in accordance with Weizmann Institute best practice (whatever that might be). Presumably, any confusion will be easy to clear up. Since WIB slices are so abundant in this offering, I am minded to cross-check them versus WIBs in other papers from the same outfit. Given their abundance, this may take some time. , 
 In Fig. 3A of this recent 2017 Cancer Research article, a STAT3 Weizmann Immunoblot slice has been flipped and reused in the adjacent slot for a different cell line. Unfortunately, this does mean that the nonequivalent phosphorylated slices directly above donu2019t amount to a hill ou2019 beans. ![image] , 
 2020 correction. https: cancerres.aacrjournals.org content 80 2 362 In the original version of this article (1), two instances of inadvertent duplication of crystal violet staining images occurred in Fig. 1D (control shRNA conditions in MDA-MB-468 and BT-20) and in Fig. 1F (0 u03bcmol L gefitinib control conditions in MDA-468 and BT-20). In addition, the same set of STAT3 Western blot bands was inadvertently duplicated in Fig. 3A (BT-20 and HCC1937). These errors have been corrected in the latest online HTML and PDF versions of the article. The authors regret these errors. Reference 1.u21b5Verma N, Mu00fcller AK, Kothari C, Panayotopoulou E, Kedan A, Selitrennik M, et al. Targeting of PYK2 synergizes with EGFR antagonists in basal-like TNBC and circumvents HER3-associated resistance via the NEDD4u2013NDRG1 axis. Cancer Res 2017;77:86u201399. ,