The emergence of tumor cells from normal precursors is thought to involve a complex interplay between genetics and cell lineage 
. Due to the different cell types involved as well as the attributes of an individual cell's local environment or niche, it is logical to assume different mechanisms are required in tumorigenesis for each lung cancer subtype. Cell lineage may also have a dramatic effect on the manifestation of genetic/epigenetic alterations during the development of each lung cancer subtype as only those promoting a malignant phenotype in the specific cellular context will be selected and maintained 
. Previous studies suggest that distinct patterns of DNA alteration exist for AC and SqCC; however, the specific genes responsible for the different tumor phenotypes are largely unknown 
In this study, we provide the first comprehensive investigation of the key genetic and epigenetic alterations distinguishing AC and SqCC lung tumors. We achieved this by integrating whole-genome DNA copy number, DNA methylation, and gene expression data to identify genes altered in a subtype-specific manner. These genes are associated with distinct gene networks in each lung cancer subtype, flagging distinct signaling pathways as contributing to tumorigenesis. We also found subtype-type specific changes to be correlated with clinical outcomes and highlight putative treatment strategies based on the subtype specific molecular signatures.
The 294 subtype-specific copy number alterations detected in this study demonstrate that different genetic pathways are involved in the pathogenesis of AC and SqCC. Importantly, previously identified lineage specific oncogenes including SOX2
were identified, validating our approach 
. Although some of the regions and genes have previously been shown to be important in NSCLC development, our findings suggest their newfound importance to a specific lung cancer subtype. For example, previously identified oncogenes such as NOTCH3
were overexpressed through increased gene dosage specifically in SqCC while the tumor suppressor KEAP1
was deleted and underexpressed specifically in AC 
. This is the first report suggesting that these previously established lung cancer-associated genes are actually involved in subtype-specific tumorigenesis.
A broader gene network-based analysis of the copy number-regulated genes revealed additional insights into the differential oncogenic mechanisms driving the pathogenesis of AC and SqCC. The top SqCC gene network was mainly associated with DNA replication, recombination and repair. In addition to these functions, histone modification genes were represented as well. Histones are fundamental building blocks of eukaryotic chromatin and are involved in myriad cellular processes, including replication, repair, recombination and chromosome segregation 
. Recently, global alterations of histone modification patterns have been reported in human cancers 
. Our data suggest that direct deregulation of histone modification enzymes including ASF1B
may drive this phenomenon and play a key role during the development of lung SqCC. As histone modifications also play an essential role in DNA replication, there may be a synergistic effect between the histone modifying genes and replication/recombination associated genes that contribute to tumor development. Interestingly, histone modification alterations occur more frequently in lung SqCC than AC, consistent with our findings 
The gene network detected as perturbed in AC subtype tumors contained genes mainly involved in regulating tissue development and cell-to-cell signaling and known to be targeted by the transcription factor HNF4α. HNF4α regulates a large set of genes in a cell-specific manner and is necessary for cell differentiation and maintenance of a differentiated epithelial phenotype 
. In other carcinomas, deregulation of HNF4α
leads to increased cellular proliferation, progression and dedifferentiation 
. This suggests that HNF4α
may act as a tumor suppressor in epithelial carcinogenesis 
. Interestingly, although HNF4α
was not affected, we found that numerous downstream targets of this gene are downregulated specifically in AC. Thus, this may have the same net affect as inactivation of HNF4α
itself and lead to increased cellular proliferation during AC tumorigenesis.
Concerted alterations to gene networks and pathways are not a feature that is limited to copy-number regulated genes. Indeed, we found that coupling subtype-specific DNA methylation profiles with matched gene expression alterations implicated numerous canonical signaling pathways in the differential development of SqCC and AC tumors. The enrichment of small-cell lung cancer signaling pathway members within the epigenetically altered SqCC genes was of particular interest. For example, one of the deregulated components of this pathway, the transcription factor E2F1,
was found to exhibit SqCC-specific hypomethylation and overexpression. E2F1
is upregulated in SCLC tumors 
, which suppresses apoptosis and induces expression of EZH2
, an oncogenic polycomb histone-methyltransferase 
. The relevance of this pathway in SqCC tumors is strengthened by our observation that EZH2
expression is significantly higher in SqCC than AC (). This is particularly interesting given the potential dual role of EZH2
in different cancer types 
. The disruption of the polycomb group (preferentially in SqCC) is relevant because we have also identified SqCC-specific deregulation of numerous histone-modifying enzymes by DNA copy number alterations.
In addition to the deregulation of histone modifying genes by DNA copy number and DNA methylation alterations, we have uncovered evidence of global SqCC-specific epigenetic disruption. Our analysis of global DNA methylation levels in AC and SqCC tumors showed that SqCC tumors were more hypomethylated overall, suggesting that the epigenetic machinery is highly deregulated in SqCC (). There is precedent for this finding, as altered global methylation is thought to be a consequence of exposure to the carcinogens found in tobacco smoke 
. Global hypomethylation, such as that caused by cigarette smoke, is also known to be associated with chromosomal instability. Although we did not observe any difference in the percentage of AC or SqCC genomes that were altered by copy number, we did identify a greater number of recurrent copy number alterations in the SqCC subtype. This may be indicative of similar selective pressures in the SqCC tumors that facilitate the development of recurrent alterations, whereas those in AC may be more diverse, leading to greater heterogeneity.
Concerted DNA copy number and DNA methylation alterations yield insight into tumor biology as well. We show hypermethylation and deletion of FHIT
to be a SqCC-specific event, confirming earlier studies describing inactivation of the gene at a higher frequency in SqCC than AC tumors 
. While there were relatively few genes that were simultaneously activated/inactivated in SqCC by DNA copy number and methylation alterations (32), there was no overlap seen in AC. In fact, compared to SqCC tumors, AC tumors possessed fewer subtype-specific alterations linked to both DNA copy number and DNA methylation. The reason for this is not clear, but it is possible that AC tumors have higher levels of cellular and/or genetic heterogeneity than SqCC tumors. Heterogeneity of patient clinical-characteristics may also contribute to this, as lung cancer in non-smokers are more likely to appear as AC tumors, and cigarette smoke may play a role in contributing to specific genetic or epigenetic alterations 
. Nevertheless, although a high proportion of our AC tumors were from never smokers (22.5%), no significant differences in copy number were identified between AC tumors from ever and never smokers (data not shown), suggesting that this is not a confounding factor in our analysis.
The specific alterations selected during the development of each subtype may also play a role in the clinical management of disease, such as influencing treatment outcomes. Indeed, genes already known to influence NSCLC response to conventional chemotherapy were deregulated in a subtype-specific manner. For example, the finding that ERCC1
disruption was subtype-specific is significant. ERCC1
is a nucleotide excision repair gene which repairs DNA adducts and lesions induced by smoking-related carcinogens 
. As such, low expression levels of ERCC1
have been implicated in lung cancer susceptibility 
and tumorigenesis, whereas high expression levels are associated with favorable overall prognosis 
. However, since ERCC1
is also involved in the repair of cisplatin-induced DNA adducts in cancer cells, high expression levels increase resistance to platinum-based chemotherapies 
, while low expression leads to drug sensitivity 
. Underscoring the relevance of this finding are the results of recent clinical trials that have described a significantly better outcome for patients who received adjuvant cisplatin-based combination chemotherapy if their resected tumors expressed low levels of ERCC1
. Our finding that this gene is inactivated specifically in AC tumors has major clinical consequences in terms of guiding disease management and treatment strategies in order to define appropriate treatment regimens for patients. This is consistent with a previous report demonstrating the subtype specificity of ERCC1
expression levels in NSCLC, and further highlights how biological differences between AC and SqCC may influence patient response to therapy 
Importantly, numerous genomic regions showed opposite patterns of alteration in each lung cancer subtype. For example, a discrete alteration spanning 2.4 Mbp on chromosome bands 8p12-p11.23 was commonly gained in SqCC and lost in AC, implying that genes in these regions may play opposite roles during the development of the individual NSCLC subtypes, acting as TSGs in AC and as oncogenes in SqCC. Such diametric alteration is seen when including epigenomic alterations as well. This is the case for PARP11
, which is upregulated in SqCC by DNA hypomethylation and downregulated in AC by copy number loss (Table S8
). This information will become particularly important as targeted therapeutic strategies based around these genes develop. The development of MEK inhibitors highlights this point 
: since activated MEK1 and MEK2 phosphorylate and activate ERK (MAPK1), the differential deregulation of MAPK1
in AC (inactivated) and SqCC (activated) tumors may be an important consideration in determining the efficacy of this treatment against lung cancer subtypes 
Similarly, numerous studies have aimed to identify genes associated with prognosis in NSCLC in order to better determine patient outcome 
. Our data suggest that these relationships may be subtype-specific as well (Table S10
). Importantly, we discovered that specific genes may be indicative of totally different clinical outcomes depending on which subtype they are disrupted in. For example, CD9
was gain/overexpressed in SqCC and high expression of this gene correlated with favorable survival in this subtype as well (). However, the opposite was true in AC, which displayed copy number loss and underexpression; low expression was associated with good survival and high expression with poor survival. Together, these results indicate that the genes involved in defining clinical characteristics are largely exclusive to individual NSCLC subtypes and influenced by the acquisition of distinct genetic alterations during tumor development. In addition, this underlines the importance of separating AC and SqCC when assessing genes involved in predicting patient prognosis and other clinical outcomes.
Furthermore, in order to demonstrate how these findings can be used to define treatment strategies tailored to the individual lung cancer subtypes, we performed CMAP analysis using our AC and SqCC specific gene signatures to identify compounds that can potentially reverse the expression of these genes. Although the results for AC were uninformative, the SqCC CMAP analysis identified numerous HDAC and PI3K/mTOR inhibitors as compounds that could potentially induce a gene expression signature negatively correlated with that associated with SqCC (Table S11
). The HDAC inhibitor result was remarkable as the alteration of histone modifying enzymes was the most prominent network disrupted in this subtype, providing a biological basis for this finding. Furthermore, cancer cells with elevated activity of E2F1 have been shown to be highly susceptible to HDAC inhibitor induced cell death and more recently HDAC inhibitors such as SAHA have been shown to suppress the activity of EZH2 
. As E2F1 and EZH2 are both upregulated in SqCC (), this data suggests that treatment with HDAC inhibitors, in conjunction with standard chemotherapy, could be a promising avenue for disease treatment. In addition, since PIK3CA
activation (mutation and/or amplification) is known to occur more frequently in SqCC than AC the finding of multiple PI3K/mTOR inhibitors as potential therapeutics for SqCC is also logical 
. Together, this data demonstrates the potential to use information about the underlying molecular biology of the cancer subtypes to make informed decisions about clinical management strategies and suggests that HDAC and PIK3/mTOR inhibitors, in combination with current treatment regimes, may provide a novel treatment tailored to lung SqCC.
Lastly, it is important to note that although they display broadly unifying characteristics, AC and SqCC themselves are very heterogeneous tumor types, with many molecular, pathologic and clinical subtypes 
. We suggest that our analysis has revealed the common initiating molecular changes for AC and SqCC, which may be followed by secondary driver mutations that cause the subsequent heterogeneity seen in advanced tumors. This is supported by the fact that we identified SqCC specific alterations in preinvasive CIS lesions, suggesting that these alterations commence early in tumor development (Table S3
). By identifying these “root” changes, one may be able to utilize type specific therapies either in combination with, or followed by, individualized therapies that target the secondary alterations to achieve a more complete antitumor response.
Fundamental discrepancies in tumor biology may be a primary factor determining the differential outcomes of lung cancer patients. Biological differences that segregate with cell lineages may also lead to differences in response to therapies 
. Therefore, tumor cell lineage may be an important consideration when selecting and developing therapeutic approaches for lung cancer. An example of this is already in common practice as SCLC and NSCLC are treated separately due to the observation that cancers of the former lineage tend to be much more responsive to initial treatment with conventional cytotoxic agents. In contrast, no clinical distinction is made between the different subtypes of NSCLC and stage is the primary factor that determine treatment options. Our high-resolution integrative analysis of NSCLC genomes and epigenomes delineated novel tumor subtype-specific genetic and epigenetic alterations responsible for driving the differential pathogenesis and phenotypes of AC and SqCC. The specific genes and networks identified in this study provide essential starting insights for elucidating mechanisms of tumor differentiation and developing tailored therapeutics for lung cancer treatment. More generally, our results confirm at the molecular level that these lung cancer subtypes are distinct disease entities. When designing new treatment strategies and testing new drugs in clinical trials, these subtype differences as well as the biological pathways should be taken into account.