Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Dex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer

Role of SnoN in Normal Epithelial Function and Tumorigenesis

The transforming growth factor-ß (TGF-ß) superfamily of cytokines regulates many cellular processes such as cell growth, cell survival, differentiation, and extracellular matrix deposition. TGF-ß is an important regulator of tissue homeostasis and is implicated in the development of human cancers and other diseases. During cancer development, TGF-ß acts as a tumor suppressor in the early stages of tumorigenesis and as a promoter of tumor invasiveness and metastasis in the later stages of cancer. TGF-ß exerts many of its diverse effects via the recruitment of the Smad proteins to activate transcription of TGF-ß target genes. Activities of the Smad proteins in the nucleus and cytoplasm have been shown to be regulated through interaction with positive and negative cellular regulators. Among the Smad negative regulators is the proto-oncogene of the Ski family SnoN, which was initially identified as a nuclear protein able to transform chicken and quail embryonic fibroblasts when overexpressed. Both pro- and anti-oncogenic activities of SnoN have been reported, but its function in normal epithelial cells has not been defined The overall purpose of the studies described in my dissertation was to elucidate the novel roles of SnoN in epithelial cell development, morphogenesis, differentiation, and tumorigenesis, using the mouse mammary gland, the non-malignant human mammary epithelial cells cultured on laminin-rich extracellular matrix (lrECM), and tissue tumor microarrays from human patients as the model systems. The work described here therefore lends insight into novel aspects of SnoN function and regulation in development and disease. SnoN expression pattern has not been well characterized and examined in normal tissues. In Chapter 3 of my dissertation, I describe the expression levels of SnoN in the mammary gland at different stages of development. SnoN is expressed at relatively low levels during puberty, but is transiently upregulated at late gestation before being downregulated during lactation and early involution. Chapter 3 describes the roles of SnoN in mammary gland development and breast cancer using our generated transgenic mice expressing a SnoN fragment under the control of the mouse mammary tumor virus promoter (MMTV). In this model system, elevated levels of SnoN increased side-branching and lobular-alveolar proliferation in virgin glands, while accelerating involution in post-lactation glands. The increased proliferation stimulated by SnoN was insufficient to induce mammary tumorigenesis. Only when cooperating with the polyoma middle T antigen (PyVmT) did SnoN accelerate the formation of aggressive multifocal adenocarcinomas and increase the formation of pulmonary metastases. Our studies define functions of SnoN in mammary epithelial cell proliferation and survival and they provide the first in vivo evidence of a pro-oncogenic role for SnoN in mammalian tumorigenesis.The roles of SnoN in epithelial differentiation and function have not been elucidated and completely understood. In Chapter 4 of my dissertation, I show that SnoN plays a very important role in maintaining mammary alveolar development, acinar structural morphogenesis, and functional differentiation of the secretory alveolar cells, using the SnoN knockout (SnoN-/-) mouse model and MCF-10A cells lacking SnoN. The impairment in alveolar development and acinar differentiation was due to a diminished prolactin-mediated STAT5 (Signal Transducer and Activator of Transcription) signaling pathway, caused by decreased STAT5 phosphorylation and total levels. While the full mechanism of SnoN and STAT5 positive regulation is still unclear, I was able to show that SnoN physcially interacts with the STAT5 protein to maintain its total levels. I also provide preliminary data showing that the high levels of SnoN observed during mammary epithelial differentiation could be regulated by both TGF-ß/Smads and prolactin/STAT5 signaling pathways. This study is the first to define novel functions of SnoN in maintaining normal mammary epithelial morphogenesis, differentiation, and function in vivo and in 3D cultures. It is also the first study to show that SnoN could regulate a different signaling pathway than TGF-ß and that SnoN expression could be regulated by a novel transcription factor different than the Smads. Evidence suggests that SnoN has both pro-oncogenic and anti-oncogenic functions by modulating both TGF-ß and p53 pathways. It is still unclear at which stages of human cancer does SnoN antagonize TGF-ß signaling to promote oncogenesis or activate p53 to induce premature senescence as a tumor suppressor mechanism. In chapter 5 of my dissertation, I examine the localization and expression levels of SnoN in tumor microarrays from patients with esophageal, ovarian, breast and pancreatic cancers. I also stain these tumors with p53 to analyze whether its inactivation in tumor tissues correlate with high levels of SnoN at different stages of tumor progression. My result suggests that SnoN levels are not overall elevated or overexpressed in the tumor samples compared to normal matched samples. However, SnoN levels were elevated in the infiltrating inflammatory and stromal cells in the advanced stages of adenocarcinomas, suggesting that SnoN might play a role in the tumor microenvironment. Finally, there was no significant correlation between high levels of SnoN and inactivation of p53 in all stages of adenocarcinomas

Expression profiles of SnoN in normal and cancerous human tissues support its tumor suppressor role in human cancer.

SnoN is a negative regulator of TGF-β signaling and also an activator of the tumor suppressor p53 in response to cellular stress. Its role in human cancer is complex and controversial with both pro-oncogenic and anti-oncogenic activities reported. To clarify its role in human cancer and provide clinical relevance to its signaling activities, we examined SnoN expression in normal and cancerous human esophageal, ovarian, pancreatic and breast tissues. In normal tissues, SnoN is expressed in both the epithelium and the surrounding stroma at a moderate level and is predominantly cytoplasmic. SnoN levels in all tumor epithelia examined are lower than or similar to that in the matched normal samples, consistent with its anti-tumorigenic activity in epithelial cells. In contrast, SnoN expression in the stroma is highly upregulated in the infiltrating inflammatory cells in high-grade esophageal and ovarian tumor samples, suggesting that SnoN may potentially promote malignant progression through modulating the tumor microenvironment in these tumor types. The overall levels of SnoN expression in these cancer tissues do not correlate with the p53 status. However, in human cancer cell lines with amplification of the snoN gene, a strong correlation between increased SnoN copy number and inactivation of p53 was detected, suggesting that the tumor suppressor SnoN-p53 pathway must be inactivated, either through downregulation of SnoN or inactivation of p53, in order to allow cancer cell to proliferate and survive. These data strongly suggest that SnoN can function as a tumor suppressor at early stages of tumorigenesis in human cancer tissues

SnoN expression in esophageal adenocarcinoma.

<p><b>A,</b> Representative SnoN staining of esophageal cancer of various grades at 20X (top) or 40X (bottom) magnifications. Two grade III samples representing different levels of SnoN expression were shown. E: epithelium; S: stroma. Green: SnoN; blue, DAPI. <b>B,</b> SnoN staining in normal and tumor epithelial cells was quantified using the Image J software and the numbers were plotted in the box plot, which includes normal samples (n = 36, mean intensity = 1.13) and esophageal tumor samples of grade I (n = 8, mean = 0.07), II (n = 19, mean = 0.71), and III (n = 11, mean = 1.26). Statistical analysis comparing the normal controls to each tumor grade showed that the epithelial SnoN levels in esophageal adenocarcinoma are significantly weaker (grade I: p = 0.0002) or similar (grade II: p = 0.1425 and grade III: p = 0.3349) to that in the control normal samples. The increase in epithelial SnoN expression in grade III compared to grade I was statistically significant (p = 0.0013)<b>. </b><b>C,</b> Quantification of SnoN stromal staining in normal samples (n = 27, mean = 1.69) and esophageal tumor samples of grade I (n = 5, mean = 0.23), II (n = 19, mean = 1.09), and III (n = 11, mean = 1.78). The statistical analysis comparing the normal controls to each esophageal tumor grade is as follow: p = 0.0023 for grade I, p = 0.8565 for II, and p = 0.1132 for grade III. The increase in stromal SnoN expression in grade II (p = 0.0287) and grade III (p = 0.0068) tumors compared to grade I tumor stroma was statistically significant.</p

SnoN is expressed in normal mammalian tissues. A,

<p>SnoN expression in the normal esophagus, including the suprabasal differentiated squamous epithelial cells, the lamina propria (stroma and connective tissue), and muscularis mucosa (smooth muscle). E: epithelial cells; F: fibroblasts; B.V; blood vessel. Negative control: tissue stained with conjugated secondary antibody alone and without primary antibody. Peptide control: tissue stained with the SnoN peptide competition control. Green: SnoN; blue, DAPI. <b>B,</b> Representative SnoN expression in the normal ovarian tissue. E: follicle epithelial cells; S: stroma. The left panel is DAPI stain alone (blue), the middle panel is SnoN stain alone (green), and the right panel is SnoN (green) plus DAPI (blue) stains. Same is true for figure panels in C-D. <b>C,</b> Representative SnoN expression in the normal pancreas. E: acinar epithelial cells; S:stromal cells of the lobular connective tissue septa. <b>D,</b> Representative SnoN expression in the normal breast. E: epithelial cells of ducts and lobuli; S: stroma.</p

SnoN expression is reduced in pancreatic adenocarcinoma samples.

<p><b>A,</b> Representative SnoN expression in the normal pancreas and in pancreatic adenocarcinoma of varying grades at 20X (top) or 40X (bottom) magnifications. E: epithelium; S: stroma. Green: SnoN; blue, DAPI. <b>B,</b> SnoN staining in normal and pancreatic tumor epithelial cells was quantified using Image J, and the numbers were plotted in the box plot, which includes normal samples (n = 5, mean = 3.08) and pancreatic tumor samples of grade I (n = 21, mean = 1.89), grade II (n = 59, mean = 1.59), and grade III (n = 8, mean = 2.09). SnoN expression in tumor samples was weaker than that in normal pancreatic samples (p = 0.0855 for grade I, p = 0.0125 for II, and p = 0.0518 for III). No significant difference was observed in SnoN epithelial staining between the pancreatic tumor samples. <b>C,</b> SnoN staining in normal (n = 2, mean = 1.87) and tumor stromal samples of grade I (n = 20, mean = 2.10), II (n = 55, mean = 1.70), and III (n = 8, mean = 1.57). There is no statistically significant difference between tumor and normal stroma samples.</p

Elevated SnoN expression correlates with inactivation of p53 in human cancer cell lines but not in primary tumor tissues.

<p><b>A,</b> 914 cancer cell lines from the Novartis CLE were classified based on their p53 gene status (lost or wild-type) as shown in the X-axis and their correlation with the copy numbers of SnoN as indicated in the Y-axis. A significant enrichment of SnoN amplification events in p53 mutant or deleted cell lines was identified (p = 7.25E-009). <b>B,</b> Cell lines from the CLE were divided into 18 different tissue lineages as depicted by various colors, and the correlation between the frequency of TP53 mutation (X-axis) and frequency of SnoN amplification (Y-axis) was determined to be highly significant with a Pearson’s correlation coefficient of 0.7. <b>C,</b> Representative p53 immunohistochemical stain in normal ovarian tissue and ovarian adenocarcinoma of grade I, grade II, and grade III (Original magnification ×20). <b>D,</b> Box plot depicting the intensity of epithelial SnoN expression (Y-axis) and p53 protein levels (as marked from 0 to 5, 0 being the lowest level in normal tissues and 5 being the highest). No significant correlation between the status of SnoN protein level and p53 inactivation was noted as measured by the Kruskal-Wallis test (p = 0.817).</p