about 85% of lung cancers are non - small - cell lung cancers ( nsclcs ) , with lung adenocarcinoma ( ladc ) and lung squamous cell carcinoma ( lscc ) as the major subtypes of nsclc.1 , 2 although encouraging targeted therapies have dramatically afforded benefits to patients with ladc , few therapeutic targets have been validated for patients with lscc.3 , 4 , 5 to date , the treatment options for patients with lscc are limited , and platinum - based chemotherapy is still the gold standard for first - line treatment ; however , some lsccs are  intrinsically resistant to chemotherapy .
virtually all patients , including initial responders , rapidly develop chemoresistance,6 , 7 , 8 , 9 which , in the absence of alternative therapies , leads to an overall 5-year survival rate of less than 20% .
therefore , a better understanding of the molecular determinants of lscc resistance to chemotherapy is a critically important step to overcome the clinical chemoresistance currently responsible for the dismal survival rates of patients with lscc .
we recently reported , for the first time , that piwi - interacting rna  likes ( pir - ls ) , a type of functional small non - coding rnas ( sncrnas ) , played critical roles in nsclc tumorigenesis and differentiation .
we have profiled pir - ls expression in normal human bronchial epithelial ( hbe ) and nsclc cells , and we found that lscc , ladc , and hbe cells could be clearly clustered together on the expression patters of all pir - ls .
we also found that pir - ls were aberrantly differentially expressed in nsclc and hbe cells as well as between lscc and ladc subtypes .
using filtered log fold change  = 1 and false discovery rate  < 0.05 as criteria , we identified that there were seven pir - ls that could distinguish lscc from both ladc and hbe cells , indicating that these seven pir - ls were lscc - related pir - ls . in this study , we observed the effects of lscc - related pir - ls in resistance caused by cisplatin ( cddp)-based chemotherapeutic agents commonly used for patients with lscc .
characteristically , we identified that pir - l-138 was upregulated upon cddp - based chemotherapy both in  vitro and in  vivo , and , further , we found that targeting the upregulated pir - l-138 resulted in enhanced sensitivity of lscc to cddp - based chemotherapy both in  vitro and in  vivo , indicating that pir - l-138 is a key contributor to cddp - based chemoresistance and that targeting pir - l-138 could be a potential strategy to overcome chemoresistance for patients with lscc .
to determine whether these lscc - related pir - ls played roles in chemoresistance , we analyzed the relationships between their expression levels and the sensitivities to gold standard first - line platinum - based chemotherapy .
given that cddp is the backbone of platinum - based chemotherapy , which is defined as a combination of a platinum compound ( cisplatin or carboplatin ) and one of the four chemotherapy agents ( gemcitabine , docetaxel , paclitaxel , and vinorelbine ) , we evaluated expression levels of lscc - related pir - ls to these agents commonly used for patients with nsclc .
we observed that lscc - related pir - ls were upregulated upon exposure to agents ( figure  s1 ) . to further identify whether these pir - ls were lscc - related sncrnas ,
we also observed both lscc - related and non - lscc - related pir - ls in four lscc as well as in four ladc cell lines ( figure  s1 ) , and pir - ls levels were measured in cells treated with cddp , gemcitabine , docetaxel , and their corresponding control solutions ( pbs or dmso ) at a drug concentration required for 50% inhibition  ( ic50 ) for 24  hr ( table s1 ; figure  s2 ) . among pir - ls , upon exposure to these chemotherapeutic agents , we observed a significant consistent and substantial increase of pir - l-138 levels in all four lscc cells treated with cddp , gemcitabine , and docetaxel ( figure  1a ) , indicating that pir - l-138 was upregulated upon cddp - based chemotherapy in  vitro .
in contrast , neither pir - l-138 in all four ladcs nor any non - lscc - related pir - ls in all four lsccs were observed to show an obviously consistent and substantial increase upon cddp , gemcitabine , or docetaxel treatment ( figure  s1 ) , indicating that the upregulated pir - l-138 was lscc - related , which was consistent with the data obtained using rna sequencing ( rna - seq ) ( figure  1b ) as we reported previously .
together , these results suggest that lscc - related pir - l-138 is upregulated upon cddp - based chemotherapy in  vitro . to determine whether the upregulated pir - l-138 upon cddp - based chemotherapy was a cell culture artifact , we analyzed its expression levels in patient - derived xenograft ( pdx ) tumors developed from tissues of patients with lscc and ladc , which were used in our previously published study .
the pdx tumors were from untreated mice or mice treated with cddp - based chemotherapy ( cddp plus gemcitabine ) for 8 and 28  days , respectively .
we observed that pir - l-138 levels were significantly increased in the tumors derived from lscc tissues ( patients umb410 and mda2131 - 11 ) at both time points after the mice had been treated with cddp - based chemotherapy for 8 and 28  days ( figures 1c and 1d ) . on the contrary ,
pir - l-138 showed an obvious downregulation in tumors derived from ladc tissues ( patient mda2131 - 1 ) at both time points after the mice had been treated with cddp - based chemotherapy for 8 and 28  days ( figure  1e ) , suggesting that pir - l-138 is induced upon cddp - based chemotherapy in tumors developed from lscc in  vivo .
next , we wanted to identify the roles of the upregulated pir - l-138 in chemoresistance of lscc . given that cddp is the backbone agent of platinum - based chemotherapy for patients with nsclc , we used cddp as the representative agent to verify roles of pir - l-138 in chemoresistance . to determine whether the cddp - induced pir - l-138 played functional roles in  vitro , we used dna oligonucleotides that were complementary to pir - l-138 as antisense ( anti-138 ) for blocking pir - l-138 because the crna oligonucleotides could trigger a short interfering rna ( sirna)-like response.10 , 12 targeting pir - l-138 followed by cddp treatment in two lscc representative cell lines of h157 and skmes-1 , we found that blocking pir - l-138 resulted in decreased cell viability ( figure  2a ) , which was caused by an increased apoptotic cell population represented by a substantial increase of the sub - g1 fragment ( figure  2b ; figures s3a 
h157 and skmes-1 cells treated with anti-138 showed a significantly increased percentage of annexin v - positive cells , cleaved capase-3 , and cleaved poly adp ribose polymerase ( parp ) ( figure  2c ; figures s4a 
s4c ) , further confirming that targeting pir - l-138 increases the sensitivity of lscc cells to cddp in  vitro . to further confirm the roles of cddp - induced pir - l-138 in apoptosis
, we used a tumor xenograft model to determine whether targeting pir - l-138 could enhance cddp - induced cell apoptosis of tumors developed from h157 cells inoculated in nu / nu mice .
h157 cells were inoculated into mice , tumors at least 5  mm in diameter were considered established , and all tumors were confirmed by pathology examination ( figure  3a , right ) .
an intraperitoneal ( i.p . ) cddp injection was given on day 1 , followed by an intratumoral ( i.t . ) injection of scr or anti-138 oligonucleotides formulated with maxsuppressor in  vivo rnalancerii on days 3 , 7 , 10 , 14 , and 16 , respectively . compared with tumors formed in untreated mice and in mice treated with cddp plus scr ,
the tumors in mice treated with cddp plus anti-138 were significantly smaller ( figure  3a , left and center and growth curves ) , indicating that targeting pir - l-138 inhibited tumor growth .
further , compared with tumors treated with cddp and scr control , tumors treated with cddp plus anti-138 exhibited higher expression of cleaved caspase 3 ( figure  3b ) , indicating that targeting pir - l-138 enhanced cddp - induced cell apoptosis in tumors .
in addition , we found that tumor areas that showed high concentrated anti-138 also showed high levels of cleaved caspase-3 and cleaved parp ( figures 3c and 3d ) , further conforming that blocking cddp - upregulated pir - l-138 accelerates cddp - induced apoptosis of tumors derived from lscc cells .
finally , we wanted to decipher the potential mechanisms underlying cddp - induced pir - l-138 in chemoresistance . given that cddp - induced pir - l-138 inhibits apoptosis and that these two lscc - representative cell lines harbor p53 mutations , we hypothesized that pir - l-138 could be involved in regulating functions of mouse double minute 2 homolog ( mdm2 ) because mdm2 and its three isoforms ( 90 , 75 , and 60  kda ) have been implicated in p53-independent apoptosis as well as in chemoresistance.15 , 16 , 17 to test our hypothesis , we measured the expression levels of total mdm2 and its cleaved isoforms after blocking and ectopic expression of pir - l-138 , respectively .
as shown in figure  4a , blocking pir - l-138 increased full - length mdm2 and decreased the 60-kda cleaved isoform ( referred to hereafter as p60-mdm2 ) upon cddp treatment , and , in contrast , ectopic expression of pir - l-138 decreased total mdm2 and increased p60-mdm2 upon cddp treatment ( figure  4b ) , indicating that the  role of pir - l-138 in apoptosis inhibition was related to p60 mdm2 .
strikingly , however , we observed that reduced p60-mdm2 was accompanied with an increase in its serine-166 phosphorylation and that increased p60-mdm2 was accompanied by a decrease in its serine-166 phosphorylation ( figures 4a and 4b ) .
based on the positive relationship between pir - l-138 and p60-mdm2 , a negative correlation with serine-166-p60-mdm2 , and our experiences with this type of sncrnas , we hypothesized that pir - l-138 could directly bind to p60-mdm2 to play roles in apoptosis . to determine
whether pir - l-138 directly interact with p60-mdm2 , biotin - conjugated pir - l-138 rna oligonucleotides were used as bait to pull down its potential binding proteins in cddp - treated h157 and skems-1 cells .
we found that p60-mdm2 was readily detectable in the pull - down products using bio - pir - l-138 but not in the products of bio - scr rna control ( figure  4c ) , indicating that pir - l-138 bound to p60-mdm2 . to confirm the interaction of pir - l-138 and p60-mdm2
, we performed an immunoprecipitation ( ip ) assay using an anti - p60-mdm2 antibody followed by rt - pcr using primers specific for pir - l-138 .
although pir - l-138 was not detectable in the ip products of a control igg antibody , pir - l-138 was easily detected in the products using the anti - p60-mdm2 antibody ( figure  4d ) . to further validate the interaction between pir - l-138 and p60-mdm2 at the individual cell level , we performed a fluorescence in  situ hybridization ( fish ) assay using a digoxin ( dig)-labeled dna probe complementary to pir - l-138 and anti - p60-mdm2 antibody .
we first tested the probes in hbe4 cells , which express a high level of pir - l-138 , to ensure the sensitivity and specificity of the pir - l-138 probe and p60-mdm2 antibody ( figures 1b and 4e ) , and we then measured their distribution upon cddp treatment .
we found that both pir - l-138 and p60-mdm2 were predominantly co - localized at the perinuclear area when unchallenged and showed an enhanced and polarized perinuclear pattern upon cddp treatment in both h157 and skmes-1 cells ( figure  4e ) .
together , these results  suggest that pir - l-138 directly interacts with p60-mdm2 in response  to cddp treatment in p53-mutated lscc h157 and skmes-1 cells .
piwi - interacting rnas ( pirnas ) have been identified to play roles in transposon silencing , heterochromatin modification , and germ cell maintenance.18 , 19 because of our limited knowledge regarding their biogenesis , most studies focused on their functions in the renewal of germline cells and development.20 , 21 , 22 , 23 their potential roles in somatic tissues , particularly in pathogenesis such as tumorigenesis , although poorly studied , have recently been recognized.10 , 18 , 19 , 24 in our recently published study , we demonstrated the presence of a type of functional sncrna class that has certain similarity with pirnas but is expressed in somatic lung bronchial epithelial cells , and we termed them pir - ls .
importantly , pir - ls not only showed differentially expressed patterns among normal hbe , lscc , and ladc cells but also played key roles in fundamental biological activities of hbe , lscc and ladc cells by directly binding to their targeted phosphorylated proteins , suggesting a biological role of pirna - ls in lung tumorigenesis and , possibly , as deterring factors of cellular behaviors for individual tumors .
because little has been studied regarding this type of sncrnas , in this study , we explored the potential effect of pir - ls in chemoresistance to the gold standard first - line platinum - based chemotherapy for treating patient with nsclc .
based on the expression levels of a pir - l that could distinguish a certain type of lung cancer from all other subtypes , we optimized a  panel of lscc - related and ladc - related pir - ls , respectively .
our initial intention was to identify an association between these lscc-/ladc - related pir - ls and sensitivity to the chemotherapeutic agents commonly used for patients with nsclc .
we first focused on lscc because few therapeutic targets are validated for patients with lscc .
we observed some relationships between pir - ls and the sensitivities to agents ; characteristically , pir - l-138 was found to be consistently and substantially upregulated after cddp - based chemotherapy treatment in lscc cell lines .
the results that pir - l-138 was significantly downregulated in lscc , could distinguish lscc from both hbe and ladc cells , and was consistently upregulated by cddp - based agents suggested that pir - l-138 might play a critical role in cddp - based chemotherapy in lscc .
it is important to note that the upregulation of pir - l-138 was not only observed in lscc cell lines but also in the pdx lscc models treated with a cddp - based regimen , indicating that the effect also occurs in lscc tissues in  vivo .
our results clearly showed that the upregulated pir - l-138 was critical in protecting lscc cells from cddp - induced apoptosis and that inhibiting pir - l-138 enhanced cddp - mediated apoptosis , suggesting targeting pir - l-138 as a strategy to boost cddp efficacy for patients with lscc .
however , a major challenge of using a nucleotide - based treatment strategy is the delivery .
our study provides a proof - of - concept result for the potential use of anti-138 to enhance cddp - mediated apoptosis of lscc cells in  vivo because we were using local injection with a sub - optimal dose , which produced only modest anti - tumor activity .
we were , therefore , focusing on determining whether anti-138 increased apoptosis at the sites of anti-138 injection by analyzing tumor tissue sections for cleaved caspase-3 and cleaved parp , which are indicators of apoptosis .
the concept was supported by the close relationship between higher levels of cleaved caspase-3 and cleaved parp and higher concentrated anti-138 ( figure  3 ) .
we identified a mechanism underlying chemoresistance caused by cddp - based chemotherapy for patients with lscc , where the functional pir - l-138 is upregulated by cddp - based agents and interacts with p60-mdm2 to inhibit apoptosis , and targeting its upregulation increases lscc sensitivity to chemotherapy agents , indicating that pir - l-138 is a key contributor to cddp - based chemoresistance and that targeting pir - l-138 could be a strategy to overcome chemoresistance for patients with lscc . in our previous study , we have shown that pir - l-163 could directly bind and regulate phosphorylated protein activities . in this study
, we explored the possibility that pir - l-138 directly interacts with the p60-mdm2 fragment upon cddp treatment in lscc cells .
the direct interaction between pir - l-138 and p60-mdm2 was demonstrated by immunoprecipitation , pir - l-138-specific pull - down , and immunofluorescence co - localization assays . although p60-mdm2 was reported to be induced in p53-wild - type,25 , 26 , 27 , 28 we identified that the upregulated pir - l-138 ,  upon cddp - based chemotherapy , inhibited cddp - induced apoptosis by interacting with the p60-mdm2 in p53-mutated lscc cells .
strikingly and interestingly , we found that there was a negative correlation between p60-mdm2 and its serine-166 phosphorylation ( figure  4a ) , indicating the possibility that the interaction of pir - l-138 and p60-mdm2 could inhibit its phosphorylation to enhance apoptosis . also , there could be a totally unknown mechanism underlying p60-mdm2 and serine-166-p60-mdm2 in regulating apoptosis by chemotherapy in p53-mutated lscc cells .
in addition , further studies are necessary to determine how pir - l-138 affects p60-mdm2 phosphorylation .
for detailed information about all cell lines used here , please refer to our previous publication .
all cell lines were recently genotyped for their authentication and routinely treated to prevent mycoplasma growth .
cell transfection was performed as described previously.10 , 29 , 30 in brief , transfection experiments were performed by using opti - mem medium and lipofectamine 3000 ( invitrogen ) for dna oligonucleotides or lipofectamine rnaimax ( invitrogen ) for rna oligonucleotides according to the manufacturer s instructions .
sncrna extraction , rt - pcr , and real - time pcr were performed as  described previously . in brief , we used adaptors for sncrna ligation , genome - specific primer ( gsp ) for reverse transcription , and specific primers for target pir - ls amplification .
an adaptor ( /5rapp/5-ctgtaggcaccatcaat-3/3ddc/ ) with both 5 and 3 modification , gsp ( 5-caagcagaagacggcatacgaattgatggtgcctacag-3 ) , a common reverse primer ( 5- caagcagaagacggcatacga-3 ) , and a pir - l-138-specific forward primer ( 5- actttagctctagaattactctgagac-3 ) were used .
the amplification conditions were as follows : denaturation at 95c for 30  s ( 5  min for the first cycle ) , annealing at 60c for 20 s , and extension at 72c for 20  s in 25 cycles for rt - pcr and in 40 cycles for real - time pcr .
a stock solution of cisplatin ( sigma ) was dissolved in pbs to a concentration of 1.7  mmol / l ( 0.5  mg / ml ) , a stock solution of docetaxel ( enzo life sciences ) was dissolved in dmso to a concentration of 10  mol / l ( 8  g / ml ) , and a stock solution of gemcitabine ( gimzar , lilly ) was dissolved in pbs to a concentration of 10  mmol / l ( 3  mg / ml ) .
cell viability and ic50 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt , life technologies ) assay as performed previously,10 , 29 , 30 and ic50 was defined as the drug concentration that was required for 50% inhibition . to avoid triggering uncertain sirna effects,10 , 12 we used complementary dna oligonucleotides as antagonists targeting pir - l-138 .
the sequence was 5-aggtctcagagtaattctagagctaaagt-3 , and the corresponding scrambled sequence was 5-gataccagggacatacgcttgatcctagc-3. transfection experiments were performed by using opti - mem medium and lipofectamine 3000 ( invitrogen ) , and the final concentration was 100  nm .
cell cycle and apoptosis analyses were performed as described previously.10 , 29 , 30 in brief , cell cycle distribution and fluorescein isothiocyanate ( fitc ) annexin v / pi ( bd pharmingen ) for the apoptosis assay were analyzed by flow cytometry ( becton dickinson ) , and dna content and percentage of apoptosis cells were analyzed on a facscan flow cytometer in combination with bd lysis software ( becton dickinson ) .
primary antibodies used were as follows : anti - cleaved parp ( 1:1,000 , clone asp214 , catalog no .
9541s , cell signaling technology ) , anti - mdm2 ( 1:200 , clone n-20 , lot l099 , catalog no .
813 , cell signaling technology ) , anti - p21 ( 1:200 , clone h-164 , catalog no .
756 , cell  signaling technology ) , anti - phospho - foxo3a ( 1:1,000 , clone ser253 , catalog no .
a228 , sigma - aldrich ) , human / mouse cleaved caspase-3 ( asp175 ) antibody ( 1:1,000 , catalog no .
31460 , pierce).10 , 11 , 29 , 30 protein extraction from cells was performed as we did before , and proteins were extracted from formalin - fixed , paraffin - embedded ( ffpe ) tissues using the qproteome ffpe tissue kit ( catalog no . 37623 , qiagen ) according to the instructions .
we followed the steps for sncrnas pull - down analysis and ip as we did previously , and the specific sequences were as follows : pir - l-138 /5biosg / aggtctcagagtaattctagagctaaagt-3control /5biosg
/ gauaccaaggacauacgcuuaugcaugcua-3. immunohistochemistry ( ihc ) was performed as we described previously.10 , 11 tumor samples were fixed in 10% neutral formalin and embedded in paraffin , and sections were prepared less than 5  mm thick , deparaffinized in xylene , and rehydrated . for h&e staining , slides were processed according to our standard protocols .
cell chamber slides ( fisher scientific ) were fixed with 4% paraformaldehyde ( pfa ) for half an hour at room temperature ( rt ) , and fissure slides were deparaffinized and rehydrated using standard protocols.10 , 11 slides were incubated with 40  g / ml protein kinase k ( roche ) at 37c for 5  min , followed by a termination reaction in 0.2% glycine ( affymetrix usb ) for 1  min before processed to pre - hybridization buffer ( 50% formamide , 1 saline - sodium citrate ( scc ) , 0.5  g / ml yeast rna , and 1 denhardt solution ) at 40c for 2  hr . probes for pir - l-138 and anti-138 were as follows : pir - l-138 : /5dign
/ actttagctctagaattactctgag-3. we added hybridization buffer ( enzo ) with dig - labeled probes and  incubated this overnight at 37c .
( 1:500 ) or cleaved caspase3 ( 1:1,000 ) at 4c overnight after washing the slides in 2 scc , 1 scc , and 0.2 scc for 10  min , followed by blocking in phosphate - buffered saline tween 20 ( pbst ) with 5% goat serum ( vector laboratories ) at rt for 2  hr .
then we incubated the slides in 2.5% goat serum with dylight 594 anti - dig ( 1:500 , catalog no .
di-7594 , vector labs ) or fitc anti - rabbit ( 1:750 , catalog no .
we sealed the slides using mounting medium with dapi for fluorescence ( vector labs ) and prepared them for scanning . for images
, we used zeiss lsm 700 for scanning the slides with the hbe4 , h157 and skmes-1 cell lines , tumor tissues slides were scanned by a cytation 3 cell imaging multi - mode reader ( biotek instruments ) and analyzed biotek s new gen5 software .
the high and low staining was distinguished by pixels of 20,000 as the cutoff , and 20 paired lesions were calculated .
tumor tissues used to determine pir - l-138 expression levels were from patient - derived nsclc xenograft models we already prepared in our previous study .
for xenograft mouse models , 5   10 h157 cells in 100  l culture medium were inoculated into the lower back and anterior chest of 6- to 8-week - old female athymic nude ( nu / nu ) mice , which are defective for development of thymic epithelium and lack cell - mediated immunity , according to the protocols approved by institutional animal care and use committees ( iacuc no.120901 ) .
the tumor sizes were measured twice a week , and tumor volumes were calculated using the formula ( /6 )  length ( l )  width ( w )  height ( h ) of tumors , where l and w represent the longest and shortest tumor dimensions , respectively . each established tumor model was confirmed by pathology examination .
cddp ( 1  mg / kg body weight / week ) injection was given on day 1 , followed by an i.t .
injection of a scramble or anti-138 dna oligo formulated with maxsuppressor in  vivo rnalancerii ( bioo scientific ) at 3  g/20  l on days 4 , 7 , 11 , and 14 , respectively .
six mice were used for each group , and the treatments lasted 16  days . statistical analysis was performed with graphpad prism 6 ( graphpad ) .
significance of the difference between groups was calculated by student s unpaired t test as we did before10 , 11 , 29 , 30 and by two - way anova ( tukey s and bonferroni s multiple comparisons test ) .
the results are reported as mean   sem , and p  < 0.05 is  considered statistically significant .
all authors reviewed , participated in revision , and approved the final version of the manuscript .
 lung cancer is the leading cause of cancer - related death worldwide . 
 although advanced drugs have benefitted patients , therapeutic success has largely been hampered because of rapid development of resistance . 
 here we report that piwi - interacting rna likes ( pir - ls ) , a novel type of functional sncrnas , play key roles in chemoresistance to cisplatin ( cddp)-based chemotherapy in lung squamous cell carcinoma ( lscc ) . 
 pir - l-138 was upregulated upon cddp - based chemotherapy both in lscc cells and in patient - derived xenograft ( pdx ) lscc models . 
 further , targeting upregulated pir - l-138 led to increased apoptosis in cddp - treated lscc cells and lscc xenograft mice treated with cddp . 
 in addition , pir - l-138 directly interacted with p60-mdm2 and inhibited cddp - activated apoptosis in p53-mutated lscc . 
 we identified the upregulated pir - l-138 upon cddp - based chemotherapy , confirmed the enhanced sensitivity of lscc to agents by targeting the upregulated pir - l-138 both in  vitro and in  vivo , and revealed mechanisms underlying pir - l-138 in chemoresistance , bolstering a new emerging clinical modality where novel functional pir - ls provide potential strategies to overcome chemoresistance for patients with lscc . 