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Supplementary Fig S9 and legend

Supplementary Figures S10-S20 and figure legends

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment.Significance:The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception.This article is highlighted in the In This Issue feature, p. 2355

Supplementary Methods

Purpose: Taxol resistance remains a major obstacle to improve the benefit of breast cancer patients. Here, we studied whether overexpression of ErbB2 may lead to mitotic deregulation in breast cancer cells via up-regulation of survivin that confers Taxol resistance.Experimental Design: ErbB2-overexpressing and ErbB2-low-expressing breast cancer cell lines were used to compare their mitotic exit rate, survivin expression level, and apoptosis level in response to Taxol. Survivin was then down-regulated by antisense oligonucleotides to evaluate its contribution to mitotic exit and Taxol resistance in ErbB2-overexpressing breast cancer cells. At last, specific PI3K/Akt and Src inhibitors were used to investigate the involvement of these two pathways in ErbB2-mediated survivin up-regulation and Taxol resistance.Results: We found that ErbB2-overexpressing cells expressed higher levels of survivin in multiple breast cancer cell lines and patient samples. ErbB2-overexpressing cells exited M phase faster than ErbB2-low-expressing cells, which correlated with the increased resistance to Taxol-induced apoptosis. Down-regulation of survivin by antisense oligonucleotide delayed mitotic exit of ErbB2-overexpressing cells and also sensitized ErbB2-overexpressing cells to Taxol-induced apoptosis. Moreover, ErbB2 up-regulated survivin at translational level and PI3K/Akt and Src activation are involved. In addition, combination treatment of Taxol with PI3K/Akt and Src inhibitor led to increased apoptosis in ErbB2-overexpressing breast cancer cells than single treatment.Conclusions: Survivin up-regulation by ErbB2 is a critical event in ErbB2-mediated faster mitotic exit and contributes to Taxol resistance.

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Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. Significance: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception. This article is highlighted in the In This Issue feature, p. 2355

Understanding cellular processes underlying early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies. While most if not all single-cell RNA sequencing (scRNA-seq) studies of lung cancer provided details on immune and stromal states, little insight is drawn to epithelial cells given their paucity (~4%) when performing unbiased scRNA-seq analysis without prior enrichment. Here, we performed in-depth scRNA-seq of enriched (by sorting for EPCAM+) epithelial cell subsets from 16 early-stage LUADs and 47 matching normal lung (NL) tissues. We also studied tissues from the same LUADs and adjacent NL by whole exome sequencing and a subset by high-resolution spatial protein and transcriptome analysis. We also performed scRNA-seq analysis of murine lungs from a human-relevant model of LUAD development following exposure to tobacco carcinogen, including strains with an alveolar type II (AT2) cell-specific lineage reporter. After extensive quality control, we retained 246,102 high quality human epithelial cells which comprised diverse normal alveolar and airway lineages as well as cancer cell populations. Diversity among cancer cells was strongly linked to LUAD oncogenic drivers. KRAS-mutant cancer cells were unique in their transcriptional features, strikingly reduced differentiation, low levels of copy number changes, and increased variability amongst the cells themselves. The local epithelial niche of LUADs, relative to that of NL, was enriched with intermediary cells in lung alveolar differentiation. A subset of these cells displayed elevated KRT8 expression (KRT8+ alveolar cells; KACs), increased plasticity and frequency of KRASG12D mutations, and its gene expression profiles were enriched in lung precancer and LUAD and signified poor survival. Notably, KACs harboring KRAS mutations were only found in the ecosystem of KRAS-mutant LUADs. Murine KACs were evident in lungs of tobacco carcinogen-exposed mice that develop KRAS-mutant LUADs but not in the saline-treated control group. While murine KACs emerged prior to tumor onset, they persisted for months after carcinogen cessation, and like their human counterparts, acquired driver Kras mutations, were poorly differentiated, and harbored KRAS-specific transcriptional programs. Spatial transcriptomics analysis showed that KAC and KRAS signatures were elevated in both murine and human tumors as well as in KACs that were in the local spatial vicinity of the LUADs. Organoids derived from lungs of tumor-bearing reporter mice were markedly enriched with KACs and were conspicuously sensitive to targeted inhibition of KRAS-G12D. This study provides new insights into the landscape of normal epithelial and malignant cells in LUAD, the role of alveolar intermediate subsets in development of the malignancy, particularly that driven by mutant KRAS, and, thus, potential targets for early interception. Citation Format: Guangchun Han, Ansam Sinjab, Warapen Treekitkarnmongkol, Zahraa Rahal, Yuejiang Liu, Alejandra G. Serrano, Jiping Feng, Ke Liang, Khaja Khan, Wei Lu, Sharia Hernandez, Xuanye Cao, Enyu Dai, Yunhe Liu, Guangsheng Pei, Jian Hu, Lorena I. Gomez Bolanos, Edwin R. Parra, Tina Cascone, Boris Sepesi, Seyed Javad Moghaddam, Paul Scheet, Marcelo V. Negrao, John V. Heymach, Mingyao Li, Jichao Chen, Steven M. Dubinett, Junya Fujimoto, Luisa M. Solis, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum Spira, Linghua Wang, Humam Kadara. An atlas of epithelial cell states and plasticity in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 113.

Understanding cellular processes underlying early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here, we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal alveolar and airway lineages as well as cancer cell populations. Diversity among cancer cells was strongly linked to LUAD patient-specific oncogenic drivers. KRAS-mutant cancer cells were unique in their transcriptional features, strikingly reduced differentiation, low levels of DNA copy number changes, and increased variability amongst the cells themselves. The local niche of LUADs, relative to that of normal lungs, was enriched with intermediary cells in lung alveolar differentiation with potential to transition to KRAS-mutant cancer cells. A subset of alveolar intermediate cells with elevated KRT8 expression (KRT8+ alveolar cells; KACs) showed increased plasticity, and their gene expression profiles were enriched in lung precancer and LUAD and signified poor survival. Murine KACs were evident in lungs of tobacco carcinogen-exposed mice that develop KRAS-mutant LUADs but not in the saline-treated control group. While murine KACs emerged prior to tumor onset, they persisted for months after carcinogen cessation, acquired driver Kras mutations and, like their human counterparts, were poorly differentiated and harbored KRAS-specific transcriptional programs. This study provides new insights into early LUAD development and identifies potential targets for treatment.

Background: Screening mammography has dramatically increased incidental detection of Ductal Carcinoma in situ (DCIS), a non-obligate precursor of breast cancer. Approximately 10-50% of DCIS progress to the invasive disease and the risk of developing recurrence or progression after surgery is still not well defined based on clinical and pathological characteristics. Our study investigated the genetic and epigenetic mechanisms associated with recurrence and progression of DCIS to invasive cancer. Methods: To identify genes associated with recurrence and progression of DCIS to invasive cancer, we performed Nanostring nCounter analysis (MDACC, n = 40) and RNA-sequencing (Sloane study, n = 183) in two independent prospective primary DCIS cohorts. Mammary epithelial-specific Pten knockout mouse model developed in our lab was used to interrogate the genetic pathway deregulated during development of mammary intraepithelial neoplasia to adenocarcinoma. ATAC-seq and ChIP-qPCR were performed to assess chromatin accessibility in the genome and validate binding of histone-modifying enzymes on target promoters. Histopathological analyses were performed to determine DCIS grades, subtypes, and protein expression profiles. Results: RNA expression profiles of normal mammary and primary DCIS tissues (n = 40) identified 10 gene transcripts that were significantly and differentially expressed between normal and high-grade DCIS. Only SOX11 expression was significantly and progressively correlated with KI67 and the DCIS recurrence score as described by Solin (JNCI 2013). Mammary intraepithelial neoplasia in Pten knockout mice and 21T DCIS to IDC progression cell line model also revealed SOX11 upregulation compared to controls. Since PTEN/PI3K/AKT pathway is known to regulate global active histone marks and open accessible chromatin, we investigated the role of AKT on SOX11 expression. Results demonstrated that inhibition of AKT altered enrichment of histone-modifying enzymes KDM5A and EZH2 on the promoter accompanying downregulated SOX11 expression in DCIS cell lines. SOX11 and EZH2 expression profiles revealed significant correlation in DCIS tissue samples. In two independent DCIS cohorts, SOX11 was found significantly upregulated in HER2+ and basal-like DCIS subtypes (P < 0.001). RNA sequencing in 183 longitudinal DCIS cases of the Sloane study (median follow-up of 96 months) revealed significantly shorter recurrence-free survival in SOX11+ than SOX11- patients (P < 0.02 in all subtypes; P = 0.002 in HER2+ subtype). Based on COX proportional hazards model, SOX11 alone was significantly associated with the risk of developing recurrence and invasive breast cancer (HR = 1.7, 95% CI = 1.1 to 2.6; P < 0.02). Conclusions: Epigenetic activation of SOX11 is associated with recurrence and progression of DCIS to invasive breast cancer. SOX11 and its epigenetic regulators can be developed as prognostic markers for DCIS patients with recurrence risk independent of traditional clinical and pathologic characteristics. Citation Format: Warapen Treekitkarnmongkol, Vandna Shah, Kazuharu Kai, Hiroshi Katayama, Justin W. Wong, Farah A Ladha, Brian Menegaz, Sarah E. Pinder, Wei Lu, Fei Yang, Ximing Tang, Savitri Krishnamurthy, Ignacio I. Wistuba, Elinor J. Sawyer, Alastair M. Thompson, Subrata Sen. Epigenetic activation of SOX11 is associated with recurrence and progression of DCIS to invasive breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr A017.

Microbial dysbiosis has emerged as a modulator of oncogenesis and response to therapy, particularly in lung cancer. Here, we investigate the evolution of the gut and lung microbiomes following exposure to a tobacco carcinogen. We performed 16S rRNA-Seq of fecal and lung samples collected prior to and at several timepoints following (nicotine-specific nitrosamine ketone/NNK) exposure in Gprc5a−/− mice that were previously shown to exhibit accelerated lung adenocarcinoma (LUAD) development following NNK exposure. We found significant progressive changes in human-relevant gut and lung microbiome members (e.g., Odoribacter, Alistipes, Akkermansia, and Ruminococus) that are closely associated with the phenotypic development of LUAD and immunotherapeutic response in human lung cancer patients. These changes were associated with decreased short-chain fatty acids (propionic acid and butyric acid) following exposure to NNK. We next sought to study the impact of Lcn2 expression, a bacterial growth inhibitor, given our previous findings on its protective role in LUAD development. Indeed, we found that the loss of Lcn2 was associated with widespread gut and lung microbiome changes at all timepoints, distinct from those observed in our Gprc5a−/− mouse model, including a decrease in abundance and diversity. Our overall findings apprise novel cues implicating microbial phenotypes in the development of tobacco-associated LUAD.

The AURKA gene, encoding Aurora kinase-A (Aurora-A), is frequently amplified and overexpressed across multiple cancer types correlating with poor prognosis. Although the AURKA gene is frequently amplified in human cancers, underlying mechanism(s) for Aurora-A protein stability through different phases of cell cycle are not well elucidated. Inhibiting the kinase activity and promoting protein degradation are two well-validated conceptual strategies for targeting protein kinases in cancers. Here, we demonstrate that Eukaryotic Elongation Factor 1 Alpha 2 (eEF1A2) facilitates PTEN-GSK3β mediated Aurora-A protein degradation through the SCF complex (SKP1-Cul1-FBXW7) during the S/G2 phase of proliferating cells. In contrast, this mechanism is inactivated in cancer cells accompanying PTEN-GSK3β pathway deficiency. Mechanistically, eEF1A2 interacts with Aurora-A, GSK3β, FBXW7 and Cul1-E3 ligase, as the SCF complex, to facilitate Aurora-A polyubiquitination for 26S proteasomal degradation. eEF1A2 promotes PTEN phosphorylation at T366 and stability, inactivates AKT and activates GSK3β which in turn phosphorylates Aurora-A at S283, S284 and S342. The phosphorylation of Aurora-A at S342 is detected during S/G2 phase of cell mitosis in parallel with eEF1A2-SCF complex formation with active form of GSK3β and neddylated Cul1. Conversely, genetic ablation of EEF1A2 and PTEN, activation of AKT, inhibition of GSK3β, expression of Aurora-A phosphodeficient-mutant attenuates the Aurora-A protein degradation which is corroborated in Aurora-A overexpressing mouse mammary carcinomas and human breast carcinomas. This study identifies a novel mechanism of Aurora-A protein degradation mediated eEF1A2-PTEN-GSK3β pathway and provides a framework for the discovery of Aurora-A therapeutic targets in breast cancer that harbors deficiency of PTEN tumor suppressor pathway. Citation Format: Treekitkarnmongkol W, Solis LM, Kai K, Thompson AM, Tian W, Wistuba II, Sasai K, Jltsumori Y, Sahin AA, Hawke DH, Lee JM, Qin L, Bawa-Khalfe T, Rad R, Wong KK, Abbott CM, Katayama H, Sen S. eEF1A2 facilitates PTEN-GSK3β mediated Aurora-A protein degradation during S-G2 phase inactivated in PTEN-deficient breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-05.

Decoding the complex molecular and cellular processes during lung adenocarcinoma (LUAD) development is needed to devise early intervention strategies. To comprehensively capture LUAD neoplastic heterogeneity and cellular plasticity, we performed single-cell RNA-sequencing (scRNA-seq) of 257,481 enriched epithelial cells (EPCAM+ sorting) from 16 early-stage LUADs, each with 3 matched normal lung (NL) samples at defined spatial proximities to the tumor (n=47). 29,076 LUAD-derived cells clustered by patient and harbored distinct gene expression features (e.g., oxidative stress response), signifying interpatient LUAD heterogeneity. We also identified, using whole exome sequencing (WES) of matching lung and germline control samples, recurrent oncogenic driver alterations (e.g., EGFR, TP53, KRAS). Transcriptomic features of malignant cells were shared between LUADs (e.g., loss of lineage-specific gene expression) or private such as those associated with driver mutation status (e.g., KRAS). Indeed, clusters of malignant cells were overall segregated based on driver mutations (e.g., KRAS, EGFR). Malignant cells from KRAS-mutant LUADs (KM-LUADs) had increased activation of NF-kB, estrogen and hypoxia signaling, comprising a unique gene module (GM) that correlated with a less differentiated state. We also found hallmark pathways (cholesterol metabolism, DNA replication, cell fate decision) specific to EGFR-mutant LUADs (EM-LUADs). Notably, cells from one EM-LUAD and its 3 multiregion NL tissues clustered closely and had activated pro-tumor lymphoid signatures (CD4 naïve, Treg). Mutation burden increased with tumor proximity and intriguingly, EGFR exon20 mutation was evident in the tumor (VAF = 0.29) and its most proximal NL (VAF = 0.05), signifying a mutational field effect. Copy number variations (CNVs) derived from WES of all samples were overall consistent with those inferred from scRNA-seq data. Relative to EM-LUADs, malignant cells from KM-LUADs displayed lower CNV burdens. Interpatient CNV heterogeneity was prominent even among LUADs harboring the same oncogenic drivers. Notably, intratumor heterogeneity (ITH) was high among epithelial cells within single regions from the same LUAD. Among LUADs, malignant cell clades with KRAS mutations and lower CNV scores displayed less differentiated states. To investigate biological pathways driving ITH, we derived 6 GMs with tumor-relevant functional features, including a transcription/translation regulation GM that consistently correlated with reduced differentiation. Our analysis of a large number of lung epithelial cells from LUAD patients reveals in-depth insights into LUAD taxonomy which can help identify epithelial heterotypes, unravel the continuum of early differentiation events and expand our understanding of early LUAD pathogenesis. Citation Format: Guangchun Han, Ansam Sinjab, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M. Solis, Edwin R. Parra, Stephen Swisher, Tina Cascone, Boris Sepesi, Jichao Chen, Steven Dubinett, Junya Fujimoto, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Single-cell sequencing of early-stage lung adenocarcinomas reveals prominent intratumoral heterogeneity and epithelial plasticity programs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2126.

Lung adenocarcinomas (LUAD) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M. Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1586.

Simple Summary The coronavirus disease 2019 (COVID-19) pandemic continues to spread rapidly on a global scale. When presenting with severe respiratory complications, COVID-19 results in markedly high death rates, particularly among patients with comorbidities such as cancer. Motivated by the ongoing global health crisis, we leveraged a growing in-house cohort of pulmonary tissues from lung cancer patients to analyze, at high resolution, the expression of host proteins implicated in the entryway of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into lung epithelial cells. Our results identify key pathways in lung pathobiology and inflammation that offer the potential to identify novel markers and therapeutic targets that can be repurposed for clinical management of COVID-19, particularly among lung cancer patients, a population that represents over half a million individuals in the United States alone. Abstract The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

Lung adenocarcinomas (LUADs) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR006.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator Lcn2 (lipocalin-2) was increased in...

Lung adenocarcinoma (LUAD) is the most commonly diagnosed histological subtype of lung cancer. While earlier work has underscored genomic and immune alterations in LUAD, the roles of individual cell populations in early-stage human LUAD evolution in space remain unknown. Here, we provide a detailed cellular atlas of early-stage LUAD and its spatial ecosystem along the peripheral lung. We performed single-cell RNA sequencing of 186,916 cells including enriched epithelial fractions from five early-stage LUADs with fourteen multi-region normal lung tissues of defined spatial proximities from the primary LUADs. We show that major epithelial and immune cellular lineages, states, and transcriptomic features geospatially and progressively evolve across normal regions and with increasing LUAD proximity. Analysis of 70,030 lung epithelial cells unraveled diverse lineage trajectories, transcriptional lineage plasticity programs underlying KRAS-mutant cells, and intratumoral heterogeneity within single sites. T regulatory cell programs including multiple immune checkpoints increased in tissues with closer proximity to LUADs, in sharp contrast to signatures of CD8+ cytotoxic T cells, antigen presentation by macrophages, and inflammatory dendritic cells. We found that some spatial signatures (e.g. a B cell signature score) were increased along the pathologic spectrum of normal lung, preneoplastic lesions, and matched invasive LUADs. LUAD cell-cell communication networks were enriched with ligand-receptor interactions involving CD24, LGALS9 and TIM3 immune checkpoints, including crosstalk between CD24 antigen in LUAD epithelial cells and SIGLEC10 in myeloid subsets. CD24 was markedly increased in preneoplasias relative to normal lung and further in LUAD, and its expression was highly positively correlated with immunosuppressive phenotypes. These data provide an atlas of cellular states and phenotypes underlying early-stage LUAD evolution in space, and a scalable resource for identification of targets for early treatment. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Kieko Hara, Patrick Brennan, Minghao Dang, Dapeng Hao, Ruiping Wang, Enyu Dai, Hitoshi Dejima, Jiexin Zhang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Kyle Chang, Danielle R. Little, Samer Bazzi, Linh Tran, Kostyantyn Krysan, Carmen Behrens, Dzifa Duose, Edwin R. Parra, Maria Gabriela Raso, Luisa M. Solis, Junya Fukuoka, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Daniel G. Rosen, John V. Heymach, Paul Scheet, Steven Dubinett, Ignacio I. Wistuba, Junya Fujimoto, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Resolving the spatial and cellular architecture of lung adenocarcinoma by multi-region single-cell sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 130.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-asociated mortality among lung cancer patients. Angiotensin converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. Yet, the single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. To fill these voids, we leveraged our ongoing efforts in single-cell transcriptomic analysis of 186,916 cells including a large number of epithelial cells (n=70,030) derived from 5 LUADs and 14 matching uninvolved normal lung tissues, to delineate expression levels and cellular distribution of ACE2 and SARS-CoV-2 priming proteases TMPRSS2 and TMPRSS4. Single-cell RNA sequencing of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2 and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, expression of TMPRSS4 was highest and most frequently detected (75%) in malignant lung cells. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. In conclusion, we describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, and that thus comprise potential treatment targets for COVID-19 particularly among lung cancer patients. Citation Format: Guangchun Han, Ansam Sinjab, Kieko Hara, Warapen Treekitkarnmongkol, Patrick Brennan, Kyle Chang, Elena Bokatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Luisa M. Solis, Boning Gao, Luc Girard, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Paul Scheet, Junya Fujimoto, Jerry Shay, John V. Heymach, John D. Minna, Steven Dubinett, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Single-cell expression landscape of SARS-CoV-2 receptor ACE2 and host proteases in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 702.

Background: The AURKA gene encoding Aurora kinase-A (Aurora-A) protein is localized on chromosome 20q13 that is frequently amplified and overexpressed across multiple cancer types correlating with patient prognosis. Aurora-A plays a pivotal role in faithful segregation of chromosomes and normal progression of mitosis, peaking at G2/M followed by degradation at the end of mitosis by APC/C (Cdh1). However, regulation of Aurora-A protein stability in human cancer cells is not well elucidated. Here, we show that Aurora-A is targeted for ubiquitination and degradation by SCF complex involving eEF1α2 and PTEN in human breast cancer cells. Methods: Using a panel of breast cancer cell lines, as in vitro models, the eEF1α2 was knocked down or ectopically expressed to test the stability of Aurora-A protein. For in vivo models, tissue micro arrays of human breast cancer were immunostained for Aurora-A and eEF1α2 expression and categorized values were statistically tested by Chi-squared test. In addition, the public breast cancer dataset (Transbig) was used to predict breast cancer prognosis by Kaplan-Meier survival analysis. Results: In breast cancer cell lines and patient samples, an eEF1α2 non-expressing group showed a trend of higher Aurora-A expression than eEF1α2 expressing group, whose trend was significant in patient samples (P Citation Format: Treekitkarnmongkol W, Kai K, Katayama H, Tian W, Rodriguez-Canales J, Sahin AA, Sen S. Aurora kinase-A protein stability is negatively regulated by eEF1α2 and PTEN in breast cancer: Prognostic and therapeutic implications [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-09.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

Lung adenocarcinoma (LUAD) is the most frequently diagnosed histological subtype of lung cancer and accounts for most smoking-related cancer deaths, warranting strategies for early intervention. Earlier work revealed genome-wide aberrations in LUADs and the adjacent premalignant field, known as “field carcinogenesis”, that are pertinent to LUAD pathogenesis. Yet, we still poorly understand the cellular and molecular architecture of LUAD and its nearby “field”. To fill this void, we performed, using the 10X Genomics system and NovaSeq 6000 platform, single-cell RNA sequencing (scRNA-seq) analysis of 4 early-stage resected LUADs as well as 11 matched normal lung tissues with differing spatial proximity from the tumors. We first analyzed 15,739 cells from a LUAD and matched tumor-adjacent and -distant normal tissues from patient 1. The resulting fraction of EPCAM+ cells from this analysis was approximately 4%. For deeper single-cell resolution of LUAD, we thus performed scRNA-seq of separately sorted/enriched epithelial cells (EPCAM+; n = 50,883) and non-epithelial cells (EPCAM-; n= 91,093) from patients 2, 3 and 4, each with an early-stage LUAD and three matched spatially-distributed normal-appearing tissues. Overall, we achieved on average approximately 150,000 reads and 1,956 genes detected per cell. Divergent populations of malignant and non-malignant cells and multiple epithelial and immune subsets clustered in an overall spatially modulated pattern according to proximity to the tumor. Hierarchical clustering revealed multiple distinct populations of airway lineage cells, including alveolar type 1 (AT1), alveolar type 2 (AT2), AT1/AT2 bipotential cells, club, goblet, basal and ciliated cells, with cells from the resected LUAD present in some but not all clusters. By analysis of epithelial-enriched fractions, we were also able to identify and interrogate rare cell subpopulations including CFTR-expressing ionocytes. By inference of copy number variation along with analysis of key oncogene expression patterns, we also identified epithelial cells that represent potential tumor cells-of-origin. Spatial reprogramming of field carcinogenesis encompassed immune cell populations including tumor-exclusive FOXP3+ regulatory T cells with marked over-expression of the major immune checkpoints CTLA4 and TIGIT. In contrast, for at least some of the cases, tumors were nearly devoid of natural killer cells, and abundance of CD8+ T cells dampened with increasing proximity to the tumor. Our single-cell surveys offer insights into novel cues in LUAD pathogenesis. Efforts are underway to interrogate the single-cell epithelial and immune landscape of additional LUADs and matched nearby normal-appearing lung, including those from non-smokers, to better understand the evolution of the disease and, thus, identify a low-hanging fruit of targets for early management of this fatal malignancy. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Patrick Brennan, Kieko Hara, Kyle Chang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Jianjun Zhang, Boris Sepesi, Tina Cascone, Don L. Gibbons, Jichao Chen, George Eapen, Edwin J. Ostrin, Junya Fujimoto, Avrum E. Spira, Paul A. Scheet, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A single-cell transcriptomic atlas of lung adenocarcinoma and adjacent normal-appearing tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1518.

Relative to other tumors in smokers, KRAS-mutant lung adenocarcinomas (LUADs) display dismal prognosis warranting the need for early management of this disease. Limiting these advances is our gap in knowledge of events that drive KRAS-mutant LUAD oncogenesis. Host defense systems, such as those elicited by the gut microbiome, were recently shown to influence tumors external to the gastrointestinal tract (e.g. melanomas), thus highlighting the microbiome as an orchestrator of oncogenesis. Yet, gut microbiome changes in early stages of KRAS-mutant LUAD development are not known. We recently reported that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-), in contrast to wild type (WT) littermates, develop LUADs which are accelerated following exposure to tobacco carcinogen (nicotine-specific nitrosamine ketone/NNK). LUADs in the tobacco exposed Gprc5a-/- mouse model exhibit high somatic mutation burdens, driver Kras variants and other co-occurring drivers, features constituting a “perfect storm” for LUAD pathogenesis in smokers. Using this human-relevant model, we performed 16S-Seq of fecal samples collected prior to and at several time points post-NNK exposure during LUAD development. We found significant, some progressive, microbial changes during the pathogenesis of LUAD, including abolishment of phylum Verrucomicrobia, progressive increase in tumor-promoting genera Odoribacter spp., gradual decrease in Akkermansia spp., which was previously shown to be associated with response to PD-1 blockade, as well as reduced abundance of Ruminococcus which was previously reported to be suppressed during colon carcinogenesis. Additionally, and prior to tumor onset, lungs and immune cells of these Gprc5a-/- mice exhibited markedly elevated expression of lipocalin 2 (LCN2), an antimicrobial protein released from host cells during microbiome imbalance and inflammation. We further found that Gprc5a-/- mice with knockout of Lcn2 exhibited increased tumors compared to similarly exposed Gprc5a-/- with intact Lcn2. These effects were accompanied by widespread changes in the gut microbiome including increased abundance of tumor-promoting Alistipes spp. and, conversely, reduced abundance of Lactobacillus spp. Our data show that host defense mediated by LCN2 counteracts Kras-mutant LUAD development by restricting gut microbiome imbalance (i.e., maintaining gut microbiome homeostasis and diversity), suggesting a protective role during Kras-mutant LUAD development. Our overall findings inform on novel pathways implicating antimicrobial host defense mechanisms in the development of smoking-associated Kras-mutant LUAD. Efforts are underway to discern specific microbiome profiles that are likely causally related to smoking-associated Kras-mutant LUAD development as well as to develop and test preclinical microbiome intervention strategies for this malignancy. Citation Format: Warapen Treekitkarnmongkol, Casey Finnicum, Ansam Sinjab, Maya Hassane, Christel Davis, Gareth E. Davies, Kristi L. Hoffman, Junya Fujimoto, Florencia McAllister, Boris Sepesi, Tina Cascone, Robert R. Jenq, Joseph Petrosino, Erik Ehli, Seyed J. Moghaddam, Humam Kadara. Evolution of the gut microbiome during the pathogenesis of smoking-associated Kras-mutant lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3349.

Relative to other lung adenocarcinomas (LUADs), Kras mutant LUAD displays dismal prognosis warranting the need for new strategies for early treatment of this lung cancer subtype. Limiting these advances is our poor understanding of events that drive Kras mutant LUAD development. In efforts to fill this void, we recently found that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-) and after tobacco carcinogen exposure (nicotine-specific nitrosamine ketone/NNK) developed LUADs harboring driver somatic Kras mutations. To understand early events preceding the onset of these Kras mutant LUADs, we performed RNA-sequencing (RNA-Seq) of normal airways in Gprc5a-/- mice and wild type (WT) littermates prior to NNK exposure. We found markedly elevated expression of the antimicrobial lipocalin 2 (Lcn2) gene in normal airways of Gprc5a-/- mice. Also, analysis of publicly available human datasets revealed elevated expression of LCN2 in human LUADs relative to normal lung tissues particularly in KRAS mutant tumors. We then sought to probe the role of Lcn2 in the pathogenesis of Kras mutant LUAD. Mice with knockout of host Lcn2 (Gprc5a-/-/Lcn2-/-) showed increased lung tumor development compared to Gprc5a-/- littermates. RNA-Seq of whole lungs at baseline and at seven months post-NNK (n = 9-10 mice each group) pinpointed differential expression profiles that denoted decreased anti-tumor immunity in Gprc5a-/-/Lcn2-/- mice. Computational estimation of immune infiltrates demonstrated increased levels of neutrophils post-NNK in Gprc5a-/-/Lcn2-/- mice but not in Gprc5a-/-littermates. By flow cytometry analysis of lung tissues, we found elevated numbers of various lymphoid and myeloid immune cells in both groups following NNK exposure and increased neutrophil counts in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- littermates. Histopathological assessment also revealed increased numbers of pro-inflammatory lesions in mice lacking Lcn2. Gprc5a-/-/Lcn2-/- mice at three months post-NNK were also found to exhibit increased production of IL-17A by lung-resident CD4+ and CD8+ T cells relative to Gprc5a-/- littermates concomitant with decreased expression of the anti-tumor Th1/cytolytic immune markers Ifnγ and Gzmb. Preliminary analysis also showed elevated expression of the major immune checkpoint PD-L1 on alveolar macrophages in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- mice with WT Lcn2. Lastly, we preliminarily found, by bacterial 16S rRNA sequencing of temporally collected fecal samples, globally altered microbiome profiles in mice lacking Lcn2. Our findings underscore novel cues in lung oncogenesis and suggest that Lcn2 decreases anti-tumor immunity, perturbs the host microbiome and promotes development of Kras mutant LUAD thereby offering new venues for early treatment of this fatal malignancy. Citation Format: Maya Hassane, Warapen Treekitkarnmongkol, Tina L. McDowell, Smruthy Sivakumar, Wenhua Lang, Joshua K. Ochieng, Sayuri Nunomura-Nakamura, Casey Finnicum, Christel Davis, Gareth E. Davies, Junya Fukuoka, Tina Cascone, Florencia McAllister, Ignacio I. Wistuba, Erik Ehli, Seyed J. Moghaddam, Paul Scheet, Junya Fujimoto, Humam Kadara. Host lipocalin 2 protects against Kras mutant lung cancer development by maintaining an anti-tumor immune contexture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4952.

Aurora kinases play critical roles in regulating spindle assembly, chromosome segregation and cytokinesis to ensure faithful segregation of chromosomes during mitotic cell division cycle. Molecular and cell biological studies have revealed that Aurora kinases, at physiological levels, orchestrate complex sequential cellular processes at distinct subcellular locations through functional interactions with its various substrates. Aberrant expression of Aurora kinases, on the other hand, cause defects in mitotic spindle assembly, checkpoint response activation and chromosome segregation leading to chromosomal instability. Elevated expression of Aurora kinases correlating with chromosomal instability is frequently detected in human cancers. Recent genomic profiling of about 3000 human cancer tissue specimens to identify various oncogenic signatures in the Cancer Genome Atlas (TCGA) project has reported that recurrent amplification and overexpression of Aurora kinase-A characterize distinct subsets of human tumors across multiple cancer types. Besides the well characterized canonical pathway interactions of Aurora kinases in regulating assembly of the mitotic apparatus and chromosome segregation, growing evidence also supports the notion that deregulated expression of Aurora kinases in non-canonical pathways drive transformation and genomic instability by antagonizing tumor suppressor and exacerbating oncogenic signaling through direct interactions with critical proteins. Aberrant expression of the Aurora kinases-p53 protein family signaling axes appears to be critical in the abrogation of p53 protein family mediated tumor suppressor pathways frequently deregulated during oncogenic transformation process. Recent findings reveal the existence of feedback regulatory loops in mRNA expression and protein stability of these protein families and their consequences on downstream effectors involved in diverse physiological functions, such as, mitotic progression, checkpoint response pathways as well as self-renewal and pluripotency in embryonic stem cells. While these investigations have focused on the functional consequences of Aurora kinase protein family interactions with wild-type p53 family proteins, those involving Aurora kinases and mutant p53 remain to be elucidated. This article presents a comprehensive review of studies on Aurora kinases-p53 protein family interactions along with a prospective view on the possible functional consequences of Aurora kinase-mutant p53 signaling pathways in tumor cells. Additionally, we also discuss therapeutic implications of these findings in Aurora kinases overexpressing subsets of human tumors.

Recent data from The Cancer Genome Atlas analysis have revealed that Aurora kinase A (AURKA) amplification and overexpression characterize a distinct subset of human tumors across multiple cancer types. Although elevated expression of AURKA has been shown to induce oncogenic phenotypes in cells in vitro, findings from transgenic mouse models of Aurora-A overexpression in mammary glands have been distinct depending on the models generated. In the present study, we report that prolonged overexpression of AURKA transgene in mammary epithelium driven by ovine β-lactoglobulin promoter, activated through multiple pregnancy and lactation cycles, results in the development of mammary adenocarcinomas with alterations in cancer-relevant genes and epithelial-to-mesenchymal transition. The tumor incidence was 38.9% (7/18) in Aurora-A transgenic mice at 16 months of age following 4-5 pregnancy cycles. Aurora-A overexpression in the tumor tissues accompanied activation of Akt, elevation of Cyclin D1, Tpx2 and Plk1 along with downregulation of ERα and p53 proteins, albeit at varying levels. Microarray comparative genomic hybridization (CGH) analyses of transgenic mouse mammary adenocarcinomas revealed copy gain of Glp1r and losses of Ercc5, Pten and Tcf7l2 loci. Review of human breast tumor transcriptomic data sets showed association of these genes at varying levels with Aurora-A gain of function alterations. Whole exome sequencing of the mouse tumors also identified gene mutations detected in Aurora-A overexpressing human breast cancers. Our findings demonstrate that prolonged overexpression of Aurora-A can be a driver somatic genetic event in mammary adenocarcinomas associated with deregulated tumor-relevant pathways in the Aurora-A subset of human breast cancer.

Metastasis remains the leading cause of cancer morbidity and mortality. ErbB2, a metastasis-promoting oncoprotein, is overexpressed in 50–60% of noninvasive ductal carcinoma in situ (DCIS). However, only 25% of invasive breast cancer (IBC) overexpress ErbB2, indicating that ErbB2 alone is not sufficient to drive metastasis and additional risk factors are necessary for the progression of ErbB2-overexpressing DCIS to IBC. A recent study published in Cancer Cell identified 14-3-3ξ as a risk factor aiding the transition of ErbB2-overexpressing DCIS into IBC. Furthermore, the study elucidated molecular mechanisms by which ErbB2 and 14-3-3ξ co-overexpression drives metastasis. Namely, ErbB2 promotes cell motility and migration via the activation of Src, while 14-3-3ξ induces epithelial-mesenchymal transition by activating TGFβ pathway to reduce cell adhesion. On the other hand, two studies recently published in British Journal of Cancer and Oncogene provide mechanistic insight into how ErbB signalling is transduced via Src, focal adhesion kinase and Ste20-like kinase to regulate focal adhesion turnover and modulate cell motility and migration. Taken together, these studies reveal that metastasis engages a variety of players that must show team spirit to win the game of spreading.

Purpose: Taxol resistance remains a major obstacle to improve the benefit of breast cancer patients. Here, we studied whether overexpression of ErbB2 may lead to mitotic deregulation in breast cancer cells via up-regulation of survivin that confers Taxol resistance. Experimental Design: ErbB2-overexpressing and ErbB2-low-expressing breast cancer cell lines were used to compare their mitotic exit rate, survivin expression level, and apoptosis level in response to Taxol. Survivin was then down-regulated by antisense oligonucleotides to evaluate its contribution to mitotic exit and Taxol resistance in ErbB2-overexpressing breast cancer cells. At last, specific PI3K/Akt and Src inhibitors were used to investigate the involvement of these two pathways in ErbB2-mediated survivin up-regulation and Taxol resistance. Results: We found that ErbB2-overexpressing cells expressed higher levels of survivin in multiple breast cancer cell lines and patient samples. ErbB2-overexpressing cells exited M phase faster than ErbB2-low-expressing cells, which correlated with the increased resistance to Taxol-induced apoptosis. Down-regulation of survivin by antisense oligonucleotide delayed mitotic exit of ErbB2-overexpressing cells and also sensitized ErbB2-overexpressing cells to Taxol-induced apoptosis. Moreover, ErbB2 up-regulated survivin at translational level and PI3K/Akt and Src activation are involved. In addition, combination treatment of Taxol with PI3K/Akt and Src inhibitor led to increased apoptosis in ErbB2-overexpressing breast cancer cells than single treatment. Conclusions: Survivin up-regulation by ErbB2 is a critical event in ErbB2-mediated faster mitotic exit and contributes to Taxol resistance.

Aurora kinase-A (referred to as Aurora-A) identified as a sereine/threonine kinase induces mitotic progression process including centrosome amplification, bipolar spindle formation and chromosome segregation through modulation of activity and localization of its interacting proteins during cell division. Beside its essential roles in mitosis, cancer profiling studies have revealed that Aurora-A function is implicated in tumor relevant signaling pathways and overexpressed in many human tumors. The findings showed highly frequent overexpression of Aurora-A in human breast ductal carcinoma in situ and primary invasive ductal breast carcinoma. Moreover, the Aurora-A overexpression correlates with poor prognosis in breast carcinoma, deregulated overexpression of Aurora-A associates with centrosome anomalies and chromosome instability in rodent models of mammary caricinogenesis. However, published studies in several transgenic mouse models attempting to address the role of Aurora-A in tumorigenesis and genomic instability have raised conflicting results. In this report we targeted expression of Aurora-A transgene in an inducible mouse model in which expression of Aurora-A was restricted to mammary epithelium during multiple pregnancy and lactation cycles under the activity of β-lactoglobulin promotor. The results demonstrate that overexpression of Aurora-A could induce tumorigenesis in mouse mammary epithelium. The tumor incidence was 70% of Aurora-A transgenic mice at 16 months of age. Of note, overexpression of Aurora-A led to genomic instability. Comparative genomic hybridization (CGH) array analyses revealed loss of Trim12a, Trim12c, Trim30b, Trim30d, Trim30e, Cdkn2d, Pten, Cep55 and gain of Adam6. Notably, Aurora-A overexpression caused nuclear accumulation of cyclin D1, activation of AKT, overexpression of TPX2 and PLK1and loss of ERα expression in tumors. Our findings indicated that prolonged Aurora-A expression in mammary epithelium leads to mammary adenocarcinomas with genomic instability. Aurora-A driven adenocarcinoma reveals deregulation of critical tumor relevant genetic pathways involving both oncogenes and tumor suppressor genes. These transgenic mouse model findings indicate that Aurora-A may be an important therapeutic target for mammary cancer. Keywords: Aurora-A, carcinogenesis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-05.

SummaryElevated Aurora kinase-A expression is correlated with abrogation of DNA damage-induced apoptotic response and mitotic spindle assembly checkpoint (SAC) override in human tumor cells. We report that Aurora-A phosphorylation of p73 at serine235 abrogates its transactivation function and causes cytoplasmic sequestration in a complex with the chaperon protein mortalin. Aurora-A phosphorylated p73 also facilitates inactivation of SAC through dissociation of the MAD2-CDC20 complex in cells undergoing mitosis. Cells expressing phosphor-mimetic mutant (S235D) of p73 manifest altered growth properties, resistance to cisplatin- induced apoptosis, as well as premature dissociation of the MAD2-CDC20 complex, and accelerated mitotic exit with SAC override in the presence of spindle damage. Elevated cytoplasmic p73 in Aurora-A overexpressing primary human tumors corroborates the experimental findings.

Cholangiocarcinoma (CCA), an adenocarcinoma of the biliary tract, is an incurable and highly lethal cancer. Its incidence has been increasing worldwide during to the past three decades and all cases have almost the same mortality rate. Due to the lack of a specific tumor marker and its silent symptom, this tumor is difficult to diagnose and the majority of patients present at the late stage (stage III or IV) of the disease progression. From 70% to 80% of CCA patients are inoperable and are generally treated by chemotherapy and radiation, which unfortunately have no significant impact for long-term survival. Thus, new diagnostic/prognosis markers and targeted therapies for CCA need to be investigated. Several lines of evidences have implicated the role of ErbB2 in aggressive forms of CCA. High expression of ErbB2 has been found in a variety of non-cancerous biliary proliferative diseases, such as in hepatolithiasis and primary sclerosing cholangitis, both of which are risk factors associated with CCA. Immunohistochemical data have demonstrated that 20% to 30% of tumor specimens from CCA patients show moderate to strong immunostaining for ErbB2 and most of them confer poor clinical outcomes (high metastasis and short survival rate). Moreover, about 30% of transgenic mice constitutively expressing wide type Neu develop gallbladder cancer and intrahepatic CCA within 8 months. Although many reports have indicated the involvement of ErbB2 in cholangiocarcinogenesis and CCA progression, the mechanism of aberrant ErbB2 expression in promoting CCA progression remains unclear. In this study, the role of ErbB2 in governing the malignant phenotype (invasion and proliferation) of CCA was investigated by suppressing ErbB2 function in three human CCA cell lines expressing different levels of ErbB2. Level of endogenous ErbB2 expression in three CCA cell lines, namely HuCCA-1, KKU-100 and KKU-M213, was determined by real time RT-PCR. Two ErbB2 inhibitory methods, a small molecule ErbB2 kinase inhibitor (AG825) and siRNA, were used to disrupt ErbB2 function in the cell lines. CCA cell invasion, motility and proliferation under ErbB2-disrupted conditions were detected using Transwell and MTT assays. In addition, ErbB2 downstream effectors were investigated by western blot analysis. Suppression of ErbB2 activity using a specific kinase inhibitor (AG825) reduced invasion, motility, and proliferation of all three CCA cell lines. The ability of this drug to inhibit neoplastic properties (invasion, motility and proliferation) increased concomitantly with the level of ErbB2 expression. Similarly, knockdown of ErbB2 level by siRNA inhibited cell invasion and proliferation of KKU-M213, a high ErbB2-expressing cell, better than those of the lower ErbB2-expressing cells, HuCCA-1 and KKU-100. Thus, both inhibitory methods indicated that there is more ErbB2-dependency for malignancy of high ErbB2-expressing cell, KKU-M213, than that of low ErbB2-expressing ones. In addition, interrupting ErbB2 activity decreased phosphorylation of AKT and p70S6K, but not ERK1/2, in the high ErbB2 expressing CCA cell line. Our data indicated that high ErbB2 expression enhances CCA invasion, motility and proliferation via AKT/p70S6K pathway, indicating the possibility of targeting these molecules for cancer therapy. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A24.