Concurrent PIK3CA mutant drives cachexia through inflammatory signaling in EGFR mutant lung cancer

Concurrent PIK3CA mutant drives cachexia through inflammatory signaling in EGFR mutant lung cancer | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Concurrent PIK3CA mutant drives cachexia through inflammatory signaling in EGFR mutant lung cancer Meiting Yue , Zhen Qin , Shijie Tang , Xinlei Cai , Yikai Zhao , Liang Chen , Luonan Chen , View ORCID Profile Hongbin Ji doi: https://doi.org/10.1101/2025.01.17.633515 Meiting Yue 1 Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences , Shanghai 200031, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhen Qin 1 Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences , Shanghai 200031, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shijie Tang 1 Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences , Shanghai 200031, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xinlei Cai 4 School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences , Hangzhou 310024, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yikai Zhao 1 Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences , Shanghai 200031, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Liang Chen 3 Institute of Life and Health Engineering, Jinan University , Guangzhou 510632, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Luonan Chen 1 Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences , Shanghai 200031, China 2 University of Chinese Academy of Sciences , Beijing 100049, China 4 School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences , Hangzhou 310024, China 5 School of Life Science and Technology, Shanghai Tech University , Shanghai 201210, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: hbji{at}sibcb.ac.cn lnchen{at}sibs.ac.cn Hongbin Ji 1 Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences , Shanghai 200031, China 2 University of Chinese Academy of Sciences , Beijing 100049, China 4 School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences , Hangzhou 310024, China 5 School of Life Science and Technology, Shanghai Tech University , Shanghai 201210, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hongbin Ji For correspondence: hbji{at}sibcb.ac.cn lnchen{at}sibs.ac.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract PIK3CA mutation is frequently concurrent with known oncogenic drivers such as EGFR mutation in lung cancer, raising an interesting question about its real function. Cachexia is a systemic disease involving complex interaction between primary tumors and distant organs, significantly contributing to cancer-related mortality. Through integrative study of genetically engineered mouse models (GEMMs) and clinical data, we find concurrent PIK3CA mutant preferentially drives cachexia in EGFR -mutant lung cancer, promoting malignant progression instead of cancer initiation. PIK3CA mutant-mediated cachexia could be overcome by osimertinib (Osi) treatment in Osi-sensitive GEMM. In contrast, chemotherapy, routinely used in clinic for those relapsed from Osi therapy, fails to ameliorate cachexia in Osi-resistant GEMM despite notable tumor suppression. PIK3CA mutant-driven cachexia is mediated through NF-κB activation and could be dampened by combined aspirin treatment. This work uncovers the biological function of PIK3CA mutant and mechanism behinds its clinical impacts, and proposes a potentially effective strategy for clinical management. Introduction PIK3CA , encoding the p110α catalytic subunit of PI3K, is mutated in approximately 7-16% of non-small-cell lung cancer (NSCLC) 1 , 2 . PIK3CA mutations are highly enriched in its helical region ( E542K , E545K ) and kinase domain ( H1047R ) 3 . It’s well established that these mutations are oncogenic and frequently cause aberrant activation of downstream AKT signaling, triggering a cascade of responses that drive lung tumorigenesis 4 . Animal model studies show that PIK3CA mutant transgene alone is sufficient to drive lung cancer initiation in mice 5 . Paradoxically, most PIK3CA mutations are found to occur concurrently with those famous oncogenic drivers 6 , 7 , e.g., EGFR mutations, KRAS mutations and ALK fusions, which are known to be mutually exclusive 8 - 11 and individually suffice to drive lung cancer initiation 12 - 14 . Previous study has shown that approximately 80% of lung cancer harboring PIK3CA mutations tends to have EGFR mutations in East Asian cohort 7 . Osimertinib (Osi), a third-generation EGFR tyrosine kinase inhibitor (TKI), is currently the preferable option for EGFR -mutant NSCLC patients, owing to its high efficacy and well-tolerated safety profile 15 . However, similar to that of early-generation EGFR-TKIs, Osi resistance inevitably develops. Additional C797S mutation represents one of the most prevalent mechanisms in Osi resistance 16 . When occurring in conjunction with a sensitizing mutation and in the absence of T790M mutation, C797S mutation confers resistance to Osi while preserving sensitivity to first- and second-generation agents. The presence of triple mutants consisting of the sensitizing mutation, T790M , and C797S , leads to resistance against all three generations of EGFR TKIs 17 . For these patients, chemotherapy, e.g., pemetrexed (PEM) in combination with cisplatin (CDDP), is the remaining option in clinic 18 . High-throughput sequencing analyses of TKI-resistant lung cancer specimens have identified the emergence of additional PIK3CA mutation 19 , 20 , indicative of its potential contribution to drug resistance. This is further supported by the observation of worse overall survival (OS) of those EGFR -mutant patients with concurrent PIK3CA mutation in their tumors 21 , 22 . However, detailed analyses of clinical TKI responses show that the concurrence of PIK3CA mutation does not affect the therapeutic efficacy of TKI treatments 21 , 22 , e.g., almost no difference of progression-free survival (PFS) in patients with or without PIK3CA mutations. This raises another interesting question regarding the real mechanism behinds the clinical impacts of PIK3CA mutant. Cancer cachexia is a complex and debilitating syndrome characterized by body weight loss primarily due to skeletal muscle atrophy and adipose tissue wasting 23 , responsible for more than 20% of cancer-related deaths 24 . Cachexia occurs in approximately 50% of lung cancer patients 25 and is associated with poor life quality 26 , elevated treatment-related toxicity 27 , 28 , reduced therapeutic responses 29 and increased risk of mortality 30 - 33 . Recent study has begun to focus on the link between specific gene alterations and cachexia development 30 . Through the integrative analyses of genetically engineered mouse models (GEMMs) and human clinical data, we here uncover an unexpected biological function of PIK3CA mutations in lung tumorigenesis, mainly contributing to cachexia instead of driving lung cancer initiation. Moreover, we also provide a reasonable explanation about the clinical observation of paradoxical difference between PFS and OS in link to concurrent PIK3CA mutation, and propose a potentially effective strategy for clinical management of lung cancer patients with concurrent PIK3CA mutation. Results PIK3CA mutant contributes to malignant progression but not lung cancer initiation To investigate the real function of PIK3CA mutant in lung tumorigenesis, we generated a genetically engineered mouse model (GEMM) by integrating the LoxP-stop-LoxP- PIK3CA E545K transgene into the Rosa26 locus of C57BL/6 mice (Fig. S1A), in which the PIK3CA mutant can be conditionally expressed via intranasal inhalation of Ad-Cre virus 34 . Unexpectedly, we found no tumor formation even after 40 weeks of Ad-Cre administration (Fig. S1B-C). This indicates that PIK3CA mutant expression alone seems insufficient to drive lung cancer initiation. Given that PIK3CA mutation and TP53 mutation frequently co-occur in human NSCLC (Fig. S1D), we further crossed PIK3CA E545K to Trp53 flox/flox mouse to see if there is any lung tumor formation. Again, we found no tumor formation in this model (Fig. S1E-F). These data collectively point to a dispensable role of PIK3CA mutant in driving lung cancer initiation. It’s well established that either EGFR mutation , KRAS mutation, or ALK fusion alone suffice in driving lung cancer initiation in mice 12 - 14 . EGFR mutation is the oncogenic driver most significantly associated with the concurrence of PIK3CA mutation (Fig. S2A). We then crossed PIK3CA E545K mice with EGFR L858R ;Trp53 flox/flox (EP) mice to generate the EGFR L858R ;PIK3CA E545K ;Trp53 flox/flox (EPP) cohort for further study (Fig. S2B). Both in vitro and in vivo assays demonstrate that PIK3CA mutant can activate the downstream AKT signaling and promotes lung cancer progression (Fig. S2C-K). Further analyses of in situ tumors showed that PIK3CA mutant significantly promoted lung cancer malignant progression, characterized by a greater degree of cellular pleomorphism and nuclear atypia 35 (Fig. S2L-M). The tumor-bearing EPP mice exhibited significantly reduced survival compared to the EP mice (Fig. S2N). These data collectively demonstrate that PIK3CA mutant mainly contributes to lung cancer malignant progression instead of driving lung cancer initiation. PIK3CA mutant drives cachexia in EGFR mutant lung cancer Interestingly, the tumor-bearing EPP mice displayed a rapid decrease in body weight starting from 6 weeks post Ad-Cre treatment, which was not observed in either wild type (WT) or EP control mice ( Fig. 1A-B ). Further analyses of the body composition revealed that the EPP mice experienced a continuous loss of lean mass along with a decrease in fat mass, aligning with the clinical diagnostic criteria for cachexia as previously established 36 (Fig. S3A-B). Moreover, fat imaging visually illustrated a significant decrease of fat tissue in these tumor-bearing EPP mice ( Fig 1C ). Compared to WT and EP mice, the EPP mice exhibited significant decrease in muscle mass in gastrocnemius (GAST), tibialis anterior (TA), and quadriceps (QUAD), as well as a decrease in epididymal white adipose tissue (eWAT) in males and the gonadal white adipose tissue (gWAT) in females ( Fig. 1D-E ). H&E staining analyses revealed the reduction of muscle fiber cross-sectional area (CSA) and adipocyte size in the EPP mice (Fig. S3C-G), histologically confirming these atrophic changes. Download figure Open in new tab Fig. 1 P I K3CA mutant is associated with cachexia development in lung cancer. A. Schematic diagram of long-term monitoring strategy of mouse body weight and body composition. B. Relative changes in body weight of WT mice (n=12), EP mice (n=12), and EPP mice (n=12) over time following intranasal delivery of Ad-Cre for 0-10 weeks. Data were normalized based on the values at the time of intranasal induction, and data collected at the 10-week post-induction were utilized for differential analysis. C. Representative fat imaging of WT mice, EP mice, and EPP mice following 8 weeks post Ad-Cre intranasal delivery. D-E. Assessed weights of the gastrocnemius (GAST), tibialis anterior (TA), quadriceps (QUAD) (D), and white adipose tissue (WAT) (E) in WT mice (n=8), EP mice (n=8), and EPP mice (n=8) following 8 weeks post Ad-Cre intranasal delivery. Data were normalized based on the average values of corresponding tissue in WT mice with the same gender. F. Average daily food intake of WT mice (n=8), EP mice (n=8), and EPP mice (n=6) following 8 weeks post Ad-Cre intranasal delivery. G-I. 24-hour activity counts (G), energy expenditure (EE) (H), and respiratory exchange ratio (RER) (I) of WT mice (n=8), EP mice (n=8), and EPP mice (n=6) following 8 weeks post Ad-Cre intranasal delivery. J. Statistical analyses of tumor burden in EP mice (n=8) and EPP mice (n=8). K-L. Assessed weights of the gastrocnemius (GAST), tibialis anterior (TA), quadriceps (QUAD) (K), and white adipose tissue (WAT) (L) in EP mice (n=8) and EPP mice (n=8). Data were normalized based on the average values of corresponding tissue in EP mice with the same gender. M. Schematic diagram of bioinformatics analysis base on TRACERx study (Al-Sawaf et al ., 2023). N. Enrichment of cachexia candidate gene list in KEGG pathways (TRACERx study, Al-Sawaf et al ., 2023). The size of the dots reflects the number of enriched genes in the pathway. The color of the dots indicates the significance of the enrichment. O. GSEA enrichment plot of PI3K-AKT signaling pathway in cancer-associated cachexia (CAC) patients compared to non-CAC patients in TRACERx study (Al-Sawaf et al ., 2023) cohort. Data are presented as mean ± SD. *P < 0.05, ***P < 0.001,****P< 0.0001 by one-way ANOVA (B, D-F), two-tailed unpaired Student’s t test (J-L). ns: not significant. Metabolic monitoring revealed that the EPP mice exhibited a significant decrease of food intake, lower level of locomotor activity, and reduced energy expenditure (EE) ( Fig. 1F-H ), all of which are hallmarks of cachexia in clinical settings 25 . Moreover, the lower respiratory exchange ratio (RER) in EPP mice indicated a metabolic shift toward the utilization of non-glucose substrates, potentially related to their decreased food intake ( Fig. 1I ). These characteristics were particularly pronounced at night, coinciding with the activity rhythms of mice (Fig. S3H-J). To exclude the potential impact of tumor burdens upon cachexia, we further analyzed the EP mice and EPP mice with comparable tumor burdens at different time points post Ad-Cre treatment, e.g., the EP mice post 17 weeks of Ad-Cre treatment and the EPP mice post 10 weeks of Ad-Cre treatment ( Fig. 1J ). Interestingly, a significant difference of cachexia features was still detectable in these two groups, e.g., the EPP mice showed lower levels of mass of skeletal muscles and adipose tissues ( Fig. 1K-L ). These data demonstrate that PIK3CA mutant triggers the cachexia symptom independent of tumor burdens. Analyses of cachexia signature proposed by TRACERx study 30 highlighted the PI3K-AKT signaling as one of the most significantly enriched pathways ( Fig. 1M-N ). Compared to patients without cachexia, the PI3K-AKT signature was also significantly enriched in those primary tumors from patients with cachexia ( Fig. 1O ). Consistently, we observed that PI3K-AKT signaling was similarly enriched in cachectic individuals across two additional datasets of lung tumor RNA sequencing (RNA-seq) 37 and serum proteomics 38 (Fig. S4A-B). These data collectively support the significant role of PIK3CA mutant in driving cachexia. Muscle wasting characteristics in EPP mice recapitulate clinical phenomena Muscle wasting is the most critical characteristic of cancer cachexia 36 . We next comparatively analyzed the gene expression profiling of TA muscles derived from WT, EP, and EPP mice ( Fig. 2A ). Principal component analysis (PCA) revealed separation between TA muscles from EPP mice and those from WT and EP mice ( Fig. 2B ). Consistently, gene expression pattern of EPP mice differed dramatically from the others ( Fig. 2C ). We found an enrichment of proteasome and autophagy signaling in TA muscles of EPP mice ( Fig. 2D ), which has been extensively documented in muscle atrophy 39 . An enrichment of features associated with muscle disorder-related diseases was also observed, suggesting that muscle atrophy in cancer cachexia may share signaling pathways with other muscle disorders. Download figure Open in new tab Fig. 2 Characteristics of muscle wasting in cachectic mice recapitulate those observed in patients with cachexia. A. Schematic diagram of murine skeletal muscle RNA sequencing. B-C. Principal component analysis (PCA) plot of tibialis anterior tissues from WT mice (n=4), EP mice (n=4), and EPP mice (n=4). C. Heatmap showing Pearson correlation coefficient (PCC) among tibialis anterior tissues from WT mice (n=4), EP mice (n=4), and EPP mice (n=4). D. Dot plot shows enriched KEGG pathways within the tibialis anterior tissues of EPP mice compared to EP mice, based on significantly differentially expressed genes (Cutoff: FDR < 0.05 and fold change ≥ 1.5). The size of the dots reflects the odds ratio of enriched genes in the pathway. The color of the dots indicates the significance of the enrichment. E. Left part contains database information; right part displays a Venn diagram showing the number of upregulated genes in muscle tissue of patients with cancer cachexia compared to non-cancer controls. F. Gene expression heatmap of tibialis anterior tissues from WT mice (n=4), EP mice (n=4), and EPP mice (n=4). We next analyzed two public databases (GSE130563 40 ; GSE133523 41 ) containing expression data from muscle tissues of cachectic patients and their respective controls. We found 42 genes upregulated in the muscles of cachectic patients, with 37 genes shared homologous counterparts in murine tissues with detectable expression ( Fig. 2E ). We further found that most of these genes were enriched in the muscle tissues of EPP mice ( Fig. 2F ), suggesting that the muscle wasting features in these mice closely resemble clinical observations. Osi effectively inhibits lung cancer progression and alleviates cachexia in TKI-sensitive EGFR L858R GEMM with concurrent PIK3CA mutant Previous studies indicated that PIK3CA mutant drives resistance to TKIs in EGFR -mutant lung cancer 19 , 20 . We then assessed the effect of single-agent Osi on mice carrying TKI-sensitive EGFR mutation ( EGFR L858R ) plus PIK3CA mutation ( Fig. 3A ). Osi treatment exhibited potent tumor-inhibitory effects, resulting in nearly complete tumor regression supported by dramatic decrease of tumor burdens and tumor numbers ( Fig. 3B-D ). In parallel, we observed increased weights in the TA, QUAD, GAST skeletal muscles, as well as in WAT tissues, indicative of the notable alleviation of cachexia ( Fig. 3E-F ). Moreover, Osi treatment mitigated atrophic changes in these tissues, as evidenced by an increase in muscle fiber CSA and adipocyte size ( Fig. 3G-K ). These data suggest that, in the TKI-sensitive GEMM, tumor malignancy and cachexia mediated by the concurrent PIK3CA mutant could be effectively suppressed by Osi treatment. Download figure Open in new tab Fig. 3 Osimertinib effectively suppresses cancer progression and alleviates cachexia in TKI-sensitive EGFR -mutant GEMM. A. Schematic diagram of osimertinib treatment strategy. B-D. Representative H&E staining images of lung tissue (B), statistical analyses of tumor burden (C) and tumor number (D) in EPP mice following gavage of either vehicle (Veh) (n=8) or 5mg/kg/day osimertinib (Osi) (n=8) for 28 days. Scale bar, 2 mm. E-F. Assessed weights of the gastrocnemius (GAST), tibialis anterior (TA), quadriceps (QUAD) (E), and white adipose tissue (WAT) (F) in EPP mice following gavage of either vehicle (Veh) (n=8) or 5mg/kg/day osimertinib (Osi) (n=8) for 28 days. Data were normalized based on the average values of corresponding tissue in mice receiving vehicle with the same gender. G-I. Representative micrographs (G), average fiber cross-sectional area (CSA) (H), and fiber CSA distribution (I) of gastrocnemius in EPP mice following gavage of either vehicle (Veh) or 5mg/kg/day osimertinib (Osi) for 28 days. Scale bar, 50 μm. J-K. Representative micrographs (J), and average size of adipocytes (K) of white adipose tissue in EPP mice following gavage of either vehicle (Veh) or 5mg/kg/day osimertinib (Osi) for 28 days. Scale bar, 50 μm. n=10 microscopic fields in each group for (H), (I) and (K). Data are presented as mean ± SD. ***P < 0.001, ****P< 0.0001 by two-tailed unpaired Student’s t test (C-F, H, K). Chemotherapy inhibits tumor growth but fails to alleviate cachexia in Osi-resistant EGFR TLCS GEMM with concurrent PIK3CA mutant Once relapses from Osi therapy, chemotherapy, e.g., PEM and CDDP, is routinely used as the conventional therapeutic strategy for patients 18 . We next developed EGFR TLCS mice carrying L858R , T790M , and C797S mutations (Fig. S5A), which confer resistance to all three generations of TKIs used in clinical practice 17 . We generated the EGFR TLCS ;Trp53 flox/flox (TLCS-P) and EGFR TLCS ;PIK3CA E545K ;Trp53 flox/flox (TLCS-PP) cohorts for further study, and found PI3K-AKT signaling was significantly enriched in mouse lung tumors derived from TLCS-PP mice (Fig. S5B). Compared to the other two groups, TLCS-PP mice showed an earlier onset of cachexia development, characterized by continuous body weight loss and reductions in both lean and fat mass ( Fig. 4A-D ). Download figure Open in new tab Fig 4. Chemotherapy fails to alleviate cachexia despite notable tumor suppression in TKI-resistant EGFR -mutant GEMM. A. Schematic diagram of long-term monitoring strategy of mouse body weight and body composition. B-D. Relative changes in body weight (B), lean mass (C), and fat mass (D) of WT mice (n=10), TLCS-P mice (n=10), and TLCS-PP mice (n=10) over time following intranasal delivery of Ad-Cre for 0-12 weeks. Data were normalized based on the values at the time of intranasal induction, and data collected at the 12-week post-induction were utilized for differential analysis. E. Schematic diagram of PEM/CDDP treatment strategy. F-G. Statistical analyses of tumor burden (F) and tumor number (G) in TLCS-PP mice following intraperitoneal injection of either vehicle (Veh) (n=5) or PEM/CDDP (n=7) for 14 days. H-I. Assessed weights of the gastrocnemius (GAST), tibialis anterior (TA), quadriceps (QUAD) (H), and white adipose tissue (WAT) (I) in TLCS-PP mice following intraperitoneal injection of either vehicle (Veh) (n=5) or PEM/CDDP (n=7) for 14 days. Data were normalized based on the average values of corresponding tissue in mice receiving vehicle with the same gender. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P< 0.0001 by one-way ANOVA (B-D), two-tailed unpaired Student’s t test (F-I). ns: not significant. We next treated the TLCS-PP mice with the standard second-line chemotherapy regimen of PEM and CDDP ( Fig. 4E ). We found chemotherapy resulted in a significant reduction of both tumor burdens and numbers, indicative of its effectiveness in suppressing tumor progression ( Fig. 4F-G ). Consistent with the adverse effects of chemotherapy on cachexia 42 , the PEM/CDDP combination failed to exert protective effect against cachexia development, as evidenced by the reduced weights of skeletal muscle and adipose tissue ( Fig. 4H-I ). These data suggest that the PIK3CA mutant-associated cachexia in Osi-resistant cases can’t be alleviated by chemotherapy despite notable tumor regression, emphasizing the need to explore the mechanisms underlying PIK3CA mutant in driving cachexia. PIK3CA mutant drives cachectic inflammation through NF- κ B activation We then performed RNA-seq on mouse lung tumors derived from both Osi-sensitive and Osi-resistant GEMMs with or without PIK3CA mutant. Enrichment of epithelial-mesenchymal transition and inflammatory response pathways has been documented as a clinical feature of primary lung tumors from cachexic patients 30 . Interestingly, we observed the enrichment of both pathways in the EPP and TLCS-PP tumors compared to their respective controls ( Fig. 5A , S5D-E). Moreover, the EPP and TLCS-PP tumors exhibited the enrichment of NF-κB signaling, which is known as the upstream regulator of multiple cachexia-associated pro-inflammatory factors 43 ( Fig. 5A , S5E). Upregulation of key factors in these tumors was further confirmed by RNA-seq and real-time PCR analyses ( Fig. 5B-D , S5F). Download figure Open in new tab Fig. 5 P I K3CA mutant increases cachexia-associated pro-inflammatory factors expression through NF- κ B activation. A. Dot plot shows Hallmark gene sets enriched in EPP tumors (n=6) versus EP tumors (n=6) based on GSEA analysis. The dot size represents the normalized enrichment score (NES). The dot color indicates enrichment significance. B-C. Gene expression heatmap of cachexia-associated pro-inflammatory factors in EP (n=6) and EPP (n=6) tumor tissues (B); as well as TLCS-P (n=3) and TLCS-PP (n=3) tumor tissues (C). D. Real-time PCR detection of mRNA levels for indicated genes in EP (n=10) and EPP (n=10) tumor tissues. E. Representative micrographs of immunofluorescence staining on EP cell line stably expressing empty vector (Ctrl) or PIK3CA E545K mutant (E545K). p65 in green, nucleus in blue (DAPI staining). Scale bar, 25 μm. F. HEK293T cells were co-transfected with NF-κB-Luc, Renilla plasmid, and either empty vector (Ctrl) or PIK3CA E545K mutant (E545K). Firefly luciferase activity was measured 48h after transfection. Firefly fluorescence were normalized based on the Renilla fluorescence values. G. Heat map displays the expression of indicated genes in the EP cell line stably expressing empty vector (Ctrl) or PIK3CA E545K mutant (E545K) with or without 10μM BAY 11L7082 treatment for 6 hours. H. Expression levels of indicated genes in non-small cell lung cancer samples with either high or low levels of PI3K-AKT signaling in The Cancer Genome Atlas (TCGA) database. I. Kaplan-Meier overall survival (OS) of non-small cell lung cancer patients with high or low cachectic inflammation signature in The Cancer Genome Atlas (TCGA) database. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P< 0.0001 by two-tailed unpaired Student’s t test (D, F), multiple t test (H), Mantel–Cox test (I). ns: not significant. To further clarify the role of NF-κB signaling in this process, we performed immunostaining analyses on the EP cells with ectopic expression of PIK3CA mutant (Fig. S2D). We found that PIK3CA mutant notably increased the nuclear translocation of p65 ( Fig. 5E ). The enhancement of NF-κB activity was further confirmed by luciferase detection ( Fig. 5F ). Additionally, cachexia-associated factors were found upregulated following ectopic expression of PIK3CA mutant and attenuated by the treatment with NF-κB inhibitor BAY 11-7082 ( Fig. 5G ). We further analyzed NSCLC patient data from The Cancer Genome Atlas (TCGA) database. Increased expression levels of cachexia-associated factors were observed in samples exhibiting elevated PI3K-AKT signaling ( Fig. 5H ). We next consolidated these factors into a cachectic inflammation signature, and found that a higher cachectic inflammation signature was associated with shorter patient overall survival ( Fig. 5I ). These data indicate that PIK3CA mutant triggers NF-κB signaling, leading to the upregulation of cachexia-associated pro-inflammatory factors, which are associated with poor prognosis of lung cancer patients. Combined aspirin treatment attenuates cachexia in EGFR mutant lung cancer with concurrent PIK3CA mutant Aspirin, a well-established non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the activity of NF-κB signaling 44 . To assess the effect of aspirin on inflammation inhibition, we treated EP tumor cells with aspirin in vitro and found that aspirin effectively suppressed the pro-inflammatory factors upregulated by ectopic expression of PIK3CA mutant (Fig. S6A). We then treated the TLCS-PP mice with aspirin alone or combined with PEM/CDDP ( Fig. 6A ). Interestingly, aspirin treatment alone significantly mitigated weight loss without notable tumor regression ( Fig. 6B-D , S6B). When combined aspirin with chemotherapy, we observed pronounced tumor regression, and the weight loss became similar to the vehicle group. Notably, we found that aspirin treatment led to increased weights in both skeletal muscle and adipose tissue, indicating effective suppression of cachexia ( Fig. 6E-F ). Aspirin also resulted in increased muscle fiber CSA and adipocyte size (Fig. S6C-F), histologically confirming the recovery from atrophic changes. These data suggest that aspirin, despite no tumor inhibition role, effectively mitigates cachexia progression of TKI-resistant lung cancer with concurrent PIK3CA mutant. Download figure Open in new tab Fig. 6 Aspirin ameliorates cachexia driven by PIK3CA mutant. A. Schematic diagram of combined treatment strategy involving PEM/CDDP and aspirin. B. Relative changes over time in body weight during intraperitoneal administration of either vehicle (Veh) (n=4), 50mg/kg aspirin (n=5), PEM/CDDP (n=6), or combined treatment (Combo) (n=6). Data were normalized based on the values at the onset of drug administration, and data collected at the 14-day time point following drug treatment were utilized for differential analysis. C-D. Representative H&E staining images of lung tissue (C) and statistical analyses of tumor burden (D) in TLCS-PP mice following intraperitoneal administration of either vehicle (Veh) (n=4), 50mg/kg aspirin (n=5), PEM/CDDP (n=6), or combined treatment (Combo) (n=6) for 14 days. Scale bar, 2 mm. E-F. Assessed weights of the gastrocnemius (GAST), tibialis anterior (TA), quadriceps (QUAD) (E), and white adipose tissue (WAT) (F) in TLCS-PP mice following intraperitoneal administration of either vehicle (Veh) (n=4), 50mg/kg aspirin (n=5), platinum-based doublet chemotherapy (PEM/CDDP) (n=6), or combined treatment (Combo) (n=6) for 14 days. Data were normalized based on the average values of corresponding tissue in mice receiving vehicle with the same gender. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA (B, D-F). ns: not significant. Discussion PIK3CA mutation has long been considered an important oncogenic driver in lung tumorigenesis, although it frequently co-occurs with EGFR mutation, KRAS mutation and ALK fusion 6 , 7 . Previous studies have convincingly shown that EGFR mutation, KRAS mutation or ALK fusion alone is sufficient to drive lung cancer initiation in mice 12 - 14 , and their co-expression even results in tumor growth inhibition 9 , 11 . This raises an interesting question about the real function of PIK3CA mutant in lung tumorigenesis. A previous study demonstrates that doxycycline-induced PIK3CA mutant transgene expression is sufficient to drive lung cancer initiation in mice 5 . In contrast, we find that Ad-Cre-mediated expression of PIK3CA mutant transgene plays a limited role in mouse lung tumor initiation. Consistently, Trejo CL et al . find no lung tumor formation in mice with endogenous expression of PIK3CA mutant 45 . No tumor is detectable in this model even after one year of PIK3CA mutant induction 46 . We reason that this discrepancy might be explained by various mouse models, the strength of PI3K pathway activation, and/or genetic backgrounds 45 . Nonetheless, clinical studies of multiple cancer types tend to support the incapability of PIK3CA mutant in driving tumor initiation. For example, human endometrial epithelium, which is pathologically normal, is found to harbor frequent PIK3CA mutations 47 . Similarly, PIK3CA mutations are detectable in pathologically normal human esophagus 48 . This study also reports no esophageal tumor detectable in PIK3CA -mutant transgenic mice 48 . These studies collectively demonstrate that PIK3CA mutant does not seem to contribute to cancer initiation as EGFR mutant, KRAS mutant, or ALK fusion does. Instead, it is more likely to play a secondary role in tumorigenesis, especially when concurrent with oncogenic drivers such as EGFR mutant, KRAS mutant, or ALK fusion. In line with this, we find that PIK3CA mutant mainly contributes to lung cancer malignant progression when coexisting with EGFR mutant, e.g., the concurrent PIK3CA mutant drives cachexia via activation of inflammatory signaling and leads to poor survival of mice bearing EGFR -mutant lung cancer. These findings provide a reasonable explanation for the longstanding paradox about the real biological function of concurrent PIK3CA mutant in lung tumorigenesis. Another paradox exists in the seemingly contradictory impact of the PIK3CA mutant upon PFS and OS of EGFR -mutant lung cancer patients following TKI therapy 21 , 22 . Eng J et al . find that concurrent PIK3CA mutation is associated with shorter survival in EGFR -mutant lung cancer; nevertheless, they find no difference of the best objective response, time to best response, time to progression, or TKI duration time between patients with and without PIK3CA mutation 22 . Similarly, Song Z et al . find that PIK3CA mutations lead to shorter OS, while these mutations do not seem to affect the PFS of EGFR -mutant patients 21 . Previous studies have observed the acquisition of PIK3CA mutations in relapsed patients after TKI resistance, indicative of its potential contribution to drug resistance, despite occurring at a low rate 19 , 20 . However, these mutations do not seem to impact the efficacy of TKIs. For example, Wu SG et al . find that the rate of PIK3CA mutation is comparable in relapsed patients vs. treatment-naïve patients, with no significant impact on therapeutic response and PFS between PIK3CA mutation-positive and -negative cases 49 . Another study also reports that 22 out of 27 EGFR -mutant patients show partial responses, including those with PIK3CA co-mutations 50 . These clinical observations point to a potentially dispensable role of PIK3CA mutant as therapeutic target. Indeed, our mouse model findings support that PIK3CA mutant contributes to lung tumorigenesis by driving cachexia rather than cancer initiation. Since PIK3CA mutant is not important for tumor initiation, tumors are less likely to rely on these mutations for survival. This explains why Osi treatment effectively inhibits the growth of EGFR -mutant lung cancer even with concurrent PIK3CA mutations. However, once patients relapse from TKI therapy and the tumors develop TKI resistance, the impact of PIK3CA mutant begins to unleash, with cachexia driven by PIK3CA mutant gradually worsening and ultimately leading to poor patient prognosis. Our data from both TKI-sensitive and -resistant EGFR -mutant mouse models collectively support this view. We find that Osi effectively inhibits both tumor growth and cachexia progression in mice bearing tumors with EGFR L858R (TKI-sensitive) and PIK3CA co-mutations. Moreover, PIK3CA-associated cachexia is unresponsive to second-line chemotherapy, as demonstrated by the reduced tissue weights in mice bearing tumors with EGFR TLCS (TKI-resistance) and PIK3CA co-mutations. This is in line with clinical observations documenting the adverse effects of chemotherapy on cachexia progression 42 . Systemic inflammation is recognized as the driving force of cachexia development in cancer 51 . We find that PIK3CA mutant activates NF-κB signaling, leading to increased expression of those cachexia-associated pro-inflammatory factors, such as IL-6 and LIF 25 . In addition to pro-inflammatory factors secreted by tumor and immune cells, chemotherapy is recognized as a significant source of systemic inflammation in the patients’ macroenvironment 52 . Recent reports demonstrate that chemotherapy induces the upregulation of pro-inflammatory cytokines and chemokines, contributing to the progression of cachexia 53 , further underscoring the significance of inflammation in cachexia progression. Lastly, we find that aspirin, a type of NSAID, effectively inhibits the progression of cachexia. Although inflammation is widely recognized as a hallmark of cachexia development, previous clinical studies indicate that targeting TNF-α, IL-6, or IL-1 individually demonstrates limited effectiveness 54 . We reason that attributing cachexia progression to a single factor is challenging, as various pro-inflammatory factors interact through complex feedback loops 51 . Therefore, broader-spectrum anti-inflammatory drugs might offer a more effective intervention. Interestingly, previous studies show that colorectal cancer patients with PIK3CA mutations receiving regular aspirin treatment exhibit improved prognosis 55 , 56 . A multicenter, multinational, prospective randomized trial recently provides the first evidence of a positive effect of adjuvant aspirin in colon cancer patients with PIK3CA mutations 57 , further emphasizing the significance of adjuvant anti-inflammatory treatment in enhancing the prognosis of these patients. Our study provides new insights into the benefit of aspirin for PIK3CA -mutant patients with colon cancer. To validate our findings across other cancer types, we further perform Gene Set Enrichment Analysis (GSEA) on a recently published pre-clinical melanoma model 58 , and observed enrichment of PI3K-AKT signaling in xenografts capable of inducing cachexia (data not shown). Consistently, a previous study also reveals an association between PIK3CA mutant and weight loss in pancreatic ductal adenocarcinoma (PDAC) patients 59 . Given the significant contribution of PIK3CA mutant to poor prognosis by driving cachexia, it will be important to further investigate its broader implications in lung cancer and beyond. Materials and methods Mouse models The Trp53 flox/flox mice were originally provided by Dr. Tyler Jacks (Cambridge, MA). The transgenic mouse models, including PIK3CA E545K , EGFR L858R , and EGFR TLCS mice were generated using CRISPR/Cas9 technology. The brief process is as follows: homologous recombination vector (donor vector) is composed of a 5′ homology arm, the indicated coding sequence (CDS), and a 3′ homology arm. Cas9 mRNA, gRNA, and the donor vector were microinjected into the fertilized eggs of C57BL/6J mice to generate the F0 generation mice. All mice were kept in specific pathogen-free environment at Shanghai Institute of Biochemistry and Cell Biology and treated in strict accordance with protocols (SIBCB-2101008) approved by the Institutional Animal Care and Use Committee of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Mice were delivered with Ad-Cre virus (2 × 10 6 p.f.u.) via nasal inhalation at 6-8 weeks of age. Osimertinib (MCE, HY-15772) was prepared in a solution containing 5% DMSO, 40% PEG300, 5% Tween-80 and 50% saline. Pemetrexed (MCE, HY-10820) or aspirin (MCE, HY-14654) was prepared in a solution containing 10% DMSO, 40% PEG300 and 50% saline. Cisplatin (MCE, HY-17394) was formulated in saline. For osimertinib treatment, EGFR L858R ;PIK3CA E545K ;Trp53 flox/flox mice were gavage daily with osimertinib (5 mg/kg/day) 6 weeks after Ad-Cre infection. Control mice were administered the vehicle solution (5% DMSO: 40% PEG300: 5% Tween-80: 50% saline). For chemotherapy and combination therapy, EGFR TLCS ;PIK3CA E545K ;Trp53 flox/flox mice were administered pemetrexed (50 mg/kg/day) combined with cisplatin (4 mg/kg/day) or aspirin (50 mg/kg/day) or both via intraperitoneal injection 10 weeks post Ad-Cre infection. Pemetrexed and cisplatin were administered weekly, and aspirin was given daily. Control mice were administered the vehicle solution (10% DMSO: 40% PEG300: 50% saline). All mice were sacrificed for gross inspection and histopathological examination. Tumor number, tumor burden, and tumor size were analyzed using ImageJ software. Cell culture and lentivirus infection HEK-293T and EP cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Cells were used for experiments within 10 to 20 passages from thawing. All cell lines were routinely tested for mycoplasma. For the establishment of stable overexpression cell line, HEK-293T cells were transfected with a 4:3:2 ratio of pCDH-CMV-PIK3CA E545K-EF1-Puro plasmid, psPAX2 plasmid (Addgene #12260), and pMD2.G plasmid (Addgene #12259). Lentiviral particles generated were then transduced into EP cells, followed by puromycin selection (3 μg/mL; Sigma-Aldrich) initiated 48 hours post-transfection and continued for an additional 2 days. Immunoblotting Whole-cell lysates of cell lines or murine tumors were generated using lysis buffer (10% SDS, 1 mM DTT, and glycerin) and incubated at 100 °C for 10 min. Equal amounts of total protein were separated by SDS-PAGE and transferred onto PVDF membranes. Protein samples were probed with specific antibodies against p110α (CST, 4249, 1:1000), AKT (CST, 9272, 1:1000), p-AKT (CST, 4070, 1:1000), S6 (CST, 2217, 1:1000), p-S6 (CST, 2215, 1:1000) and β-actin (Abclonal, AC026, 1:50000). Protein expression was assessed by Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and detected on SAGECREATION (Sage Creation Science Co.). RNA Isolation, reverse transcription, and real-time PCR Total RNA from cultured cells or tissue samples was extracted using TRIzol (Invitrogen), and complementary DNA was synthesized from 1 μg of RNA using the PrimeScript RT Reagent Kit (TaKaRa). Real-time PCR was performed on a LightCycler ® 96 System (Roche) using SYBR Green I Master (Roche). β-actin was utilized as the internal control. The following primers were used: Il1b F: 5’-GCAACTGTTCCTGAACTCAAC-3’, R: 5’-ATCTTTTGGGGTCCGTCAACT-3’. Il6 F: 5’-TCTGCAAGAGACTTCCATCCAGTTGC-3’, R: 5’-AGCCTCCGACTTGTGAAGTGGT-3’. Il11 F: 5’-CTGACGGAGATCACAGTCTGGA-3’, R: 5’-GGACATCAAGTCTACTCGAAGCC-3’. Lif F: 5’-AAAAGCTATGTGCGCCTAACA-3’, R: 5’-GTATGCGACCATCCGATACAG-3’. Osm F: 5’-ACGGTCCACTACAACACCAG-3’, R: 5’-CCATCGTCCCATTCCCTGAAG-3’. Ptgs2 F: 5’-TTCAACACACTCTATCACTGGC-3’, R: 5’-AGAAGCGTTTGCGGTACTCAT-3’. Tnf F: 5’-CTGAACTTCGGGGTGATCGG-3’, R: 5’-GGCTTGTCACTCGAATTTTGAGA-3’. Actb F: 5’-GGCTGTATTCCCCTCCATCG-3’, R: 5’-CCAGTTGGTAACAATGCCATGT-3’. Soft agar colony formation assay Soft agar assay was performed as previously described 60 . In detail, a bottom layer of 1% agar with complete medium was first solidified. An upper layer was then added, containing 500 cells suspended in a 0.4% agar medium mixture in 6-well plates. After 2–3 weeks of incubation, cells were stained with 0.005% crystal violet, and the number of colonies was counted. Immunofluorescence analyses Cells grown on glass coverslips were washed with cold PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 15Lmin at room temperature. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 15Lmin. After blocking with 4% bovine serum albumin (BSA) in PBS for 1Lh, cells were probed with p65 (CST, 8242, 1:500) overnight at 4°C. After washing three times with PBST, secondary antibodies were added and incubated for 1 h at room temperature. After washed with PBST, the coverslips were mounted onto glass slides using fluorescent mounting medium. Confocal images were captured using a Leica TCS SP8 system with a HC PL APO CS2 63×/1.40 oil objective. Luciferase reporter gene assay HEK293T cells were seeded in 12-well plate at 3x10 5 cells/well. 24 h after seeding, cells were transiently transfected with 1 μg of either PIK3CA E545K plasmid or the control vector, along with 1 μg NF-κB-Luc plasmid and 500 ng Renilla plasmid. 48 hours post-transfection, cells were harvested, and the Dual-Luciferase Reporter Assay System (Promega) was used for detection. Tissue collection Mice were euthanized, and their body weight was recorded. The lungs with tumors were excised and fixed in 4% formalin; a portion of the tumors was snap-frozen for further analysis. Gastrocnemius (GAST), tibialis anterior (TA), quadriceps (QUAD), epididymal white adipose tissue (eWAT), and gonadal white adipose tissue (gWAT) were dissected and weighed. The white adipose tissues were fixed in 4% formalin for histological examination. One part of the skeletal muscle was snap-frozen for RNA-seq, while the remaining portion was embedded in optimal cutting temperature (OCT) compound for rapid freezing and subsequent histological examination. Body composition analysis and fat imaging Mice were weighed and body composition was measured using Bruker’s minispec LF50 Body Composition Analyzer. The instrument provides measurements of three related components: fat, free body fluids, and lean tissue mass. The imaging of mouse adipose tissue was performed using the NM42-060H-I (Niumag) small animal magnetic resonance imaging (MRI) scanner. Metabolic cage Mice were individually housed in CLAMS metabolic cages (Columbus Instruments) for a duration of 3 days. Oxygen consumption (VO2) and carbon dioxide expiration (VCO2) were measured for 1 min with 14 min intervals at a flow rate set at 0.72 liter per minute. Respiratory exchange ratio (RER) was calculated as the ratio of VCO2 to VO2. Simultaneously, locomotor activity and energy expenditure were recorded using the built-in detection system, while food intake was manually measured at the same designated time each day. RNA-seq analyses Raw fastq data from RNA-seq were processed with Trimmomatic 61 (v0.39) for adapter trimming and low-quality read filtering. The processed data were then aligned to the mm10 reference genome using STAR 62 (v2.5.2b). Genes with zero expression in more than 70% of the samples were filtered out. FPKM normalization and log2 transformation were applied to the raw count data, followed by differential expression analysis using limma 63 to identify differentially expressed genes (DEGs) between conditions. Pathway enrichment analysis was conducted using the Enrichr 64 method based on the resulting DEGs. In addition, GSEA enrichment analysis was performed with the clusterProfiler 65 package based on ranked genes. Statistical analyses For comparing means of two groups, differences were analyzed by Student’s t test (two-tailed) and performed by Prism GraphPad software. For comparing means of three or more than three groups, differences among groups were analyzed by one-way ANOVA performed by Prism GraphPad software. P value <0.05 was considered statistically significant. Error bars were represented with SD. Survival analysis was performed using the Kaplan–Meier method. Author Contributions H.J. conceived the idea and designed the experiments. M.Y. and Z.Q. performed all experiments and analyzed the data. S.T. and X.C. performed the bioinformatics analyses. Y.Z. established primary cell line. L.C. and L.C. provided technical assistance and helpful comments. H.J. and M.Y. wrote the manuscript. All authors approved the final version. Illustration Tool The graphical abstract image is created with BioRender. Disclosure of conflicts of interest The authors declare no potential conflicts of interest. Fig. S1 PIK3CA mutant shows limited tumor initiation capacity in lung cancer. A. Schematic diagram of LoxP-stop-LoxP- PIK3CA E545K mouse model. B. Representative H&E staining images of lung tissue from PIK3CA E545K mice following 40 weeks post Ad-Cre intranasal delivery. Scale bar, 2 mm. C. Representative micrographs of PIK3CA E545K mice following 40 weeks post Ad-Cre intranasal delivery. Scale bar, 50 μm. D. Mutation status and co-occurrence analysis of PIK3CA and TP53 in non-small cell lung cancer patients from the cBioPortal cancer genomics database. E. Representative H&E staining images of lung tissue from PIK3CA E545K ;Trp53 flox/flox mice following 40 weeks post Ad-Cre intranasal delivery. Scale bar, 2 mm. F. Representative micrographs of PIK3CA E545K ;Trp53 flox/flox mice following 40 weeks post Ad-Cre intranasal delivery. Scale bar, 50 μm. Fig. S2 PIK3CA mutant promotes murine lung cancer malignant progression. A. Mutation status and co-occurrence analysis of PIK3CA, EGFR, KRAS, BRAF, and MAP2K1 in non-small cell lung cancer patients from the cBioPortal cancer genomics database. B. Schematic diagram of LoxP-stop-LoxP- EGFR L858R mouse model. C. Schematic diagram of EP cell line establishment. D. Immunoblotting analysis of phosphorylated AKT and phosphorylated S6 in EP cell line. E-G, Representative images of soft agar colony formation (E), statistical analyses of colony numbers (F), and average colony size (G) of EP cell line stably expressing empty vector (Ctrl) or PIK3CA E545K mutant (E545K). Scale bar, 500 μm. H. Immunoblotting analysis of phosphorylated AKT and phosphorylated S6 in EGFR L858R ; Trp53 -/- (EP) and EGFR L858R ; PIK3CA E545K ; Trp53 -/- (EPP) in situ tumor tissues. I-J. Statistical analyses of in situ tumor burden (I) and tumor number (J) in EP mice and EPP mice following 6 weeks (n=3), 8 weeks (n=3), and 10 weeks (n=5) post Ad-Cre intranasal delivery. K. Statistical analyses of individual size of EP tumor (n=160) and EPP tumor (n=178) following 10 weeks post Ad-Cre intranasal delivery. L. Statistical analyses of in situ tumor grade in EP mice (n=5) and EPP mice (n=5) following 10 weeks post Ad-Cre intranasal delivery. M. Representative micrographs of in situ tumors with different grading characteristics in EP and EPP mice following 10 weeks post Ad-Cre intranasal delivery. Scale bar, 50 μm. N. Kaplan-Meier curve shows the overall survival of EP mice (n=6), and EPP mice (n=6) receiving Ad-Cre delivery. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P< 0.0001 by two-tailed unpaired Student’s t test (F, G, K), multiple t test (I-J). ns: not significant. Fig. S3 PIK3CA mutant promotes lung cancer cachexia progression. A-B. Relative changes in lean mass (A), and fat mass (B) of WT mice (n=12), EP mice (n=12), and EPP mice (n=12) over time following intranasal delivery of Ad-Cre for 0-10 weeks. Data were normalized based on the values at the time of intranasal induction, and data collected at the 10-week post-induction were utilized for differential analysis. C-E. Representative micrographs (C), average fiber cross-sectional area (CSA) (D), and fiber CSA distribution (E) of gastrocnemius tissue in WT mice, EP mice, and EPP mice following 8 weeks post Ad-Cre intranasal delivery. Scale bar, 50 μm. F-G. Representative micrographs (F), and average size of adipocytes (G) of white adipose tissue in WT mice, EP mice, and EPP mice following 8 weeks post Ad-Cre intranasal delivery. Scale bar, 50 μm. H-J. Statistical analyses of activity counts (H), energy expenditure (EE) (I), and respiratory exchange ratio (RER) (J) of WT mice (n=8), EP mice (n=8), and EPP mice (n=6) following 8 weeks post Ad-Cre intranasal delivery. n=10 microscopic fields in each group for (D), (E) and (G). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P< 0.0001 by one-way ANOVA (A-B, E, G, H-J). Fig. S4 PI3K-AKT activation is associated with cachexia development in lung cancer patients. A. GSEA enrichment plot of PI3K-AKT signaling pathway in low-muscularity (LM) patients compared to high-muscularity (HM) patients in Cury et al . cohort. B. Dot plot shows enriched KEGG pathways in the serum proteomics of cachexia patients compared to non-cachexia patients in Wang et al . cohort. Fig. S5 Gene expression characterization of murine tumors with PIK3CA mutations. A. Left part shows the schematic diagram of LoxP-stop-LoxP- EGFR TLCS mouse model; right part shows the representative H&E staining images of lung tissue from EGFR TLCS mice post Ad-Cre intranasal delivery. Scale bar, 2 mm. B-E. GSEA enrichment plot of PI3K-AKT signaling pathway (B), inflammatory response (C), epithelial mesenchymal transition (D), and Tnf-α signaling via NF-κB pathway (E) in TCLS-PP tumors compared to TLCS-P tumors. F. Real-time PCR detection of mRNA levels for indicated genes in TLCS-P (n=12) and TLCS-PP (n=12) tumor tissues. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired Student’s t test (F). Fig. S6 Aspirin effectively ameliorates cachexia in PIK3CA -mutant lung cancer. A. Heat map of indicated genes expression in EP cell line stably expressing empty vector (Ctrl) or PIK3CA E545K mutant (E545K) with or without 2mM aspirin treatment for 24 hours. B. Statistical analyses of tumor number in TLCS-PP mice following intraperitoneal administration of either vehicle (Veh) (n=4), 50mg/kg aspirin (n=5), PEM/CDDP (n=6), or combined treatment (Combo) (n=6) for 14 days. C-D. Representative micrographs (C), and average fiber cross-sectional area (CSA) (D) of gastrocnemius tissue in TLCS-PP mice following intraperitoneal administration of either vehicle (Veh), 50mg/kg aspirin, PEM/CDDP, or combined treatment (Combo) for 14 days. Scale bar, 50 μm. E-F. Representative micrographs (E), and average size of adipocytes (F) of white adipose tissue in TLCS-PP mice following intraperitoneal administration of either vehicle (Veh), 50mg/kg aspirin, PEM/CDDP, or combined treatment (Combo) for 14 days. Scale bar, 50 μm. n=10 microscopic fields in each group for (D) and (F). Data are presented as mean ± SD. *P < 0.05, ****P< 0.0001 by one-way ANOVA (B, D, F). ns: not significant. Acknowledgments We thank Dr. Tyler Jacks and Dr. Liang Chen for providing the mice. This work was supported by the National Key Research and Development Program of China (grants 2022YFA1103900 to H.J.; 2020YFA0803300 to H.J.); the National Natural Science Foundation of China (grants 82303039 to Z.Q., 82341002 to H.J., 32293192 to H.J., 82030083 to H.J.); the Shanghai Sailing Program(23YF1452900 to Z.Q.); the Basic Frontier Scientific Research Program of Chinese Academy of Science (ZDBS-LY-SM006 to H.J.); the Innovative research team of high-level local universities in Shanghai (SSMU-ZLCX20180500 to H.J.). References 1. ↵ Cancer Genome Atlas Research, N . Comprehensive molecular profiling of lung adenocarcinoma . Nature 511 , 543 - 50 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ Cancer Genome Atlas Research, N . Comprehensive genomic characterization of squamous cell lung cancers . Nature 489 , 519-25 ( 2012 ). OpenUrl PubMed 3. ↵ Arafeh , R. & Samuels , Y . 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Share Concurrent PIK3CA mutant drives cachexia through inflammatory signaling in EGFR mutant lung cancer Meiting Yue , Zhen Qin , Shijie Tang , Xinlei Cai , Yikai Zhao , Liang Chen , Luonan Chen , Hongbin Ji bioRxiv 2025.01.17.633515; doi: https://doi.org/10.1101/2025.01.17.633515 Share This Article: Copy Citation Tools Concurrent PIK3CA mutant drives cachexia through inflammatory signaling in EGFR mutant lung cancer Meiting Yue , Zhen Qin , Shijie Tang , Xinlei Cai , Yikai Zhao , Liang Chen , Luonan Chen , Hongbin Ji bioRxiv 2025.01.17.633515; doi: https://doi.org/10.1101/2025.01.17.633515 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cancer Biology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17648) Bioengineering (13871) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18561) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22475) Immunology (17713) Microbiology (40328) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)