Gloria H. Su

mTOR and P70 S6 Kinase Expression in Primary Liver Neoplasms

Purpose: mTOR and P70 S6 kinase (S6K) play a key role in regulating protein translation. The role of mTOR and S6K in hepatocellular carcinoma has not been investigated, but this pathway is of particular interest because an effective inhibitor, rapamycin, is available. This study was undertaken to determine the prevalence and clinicopathological correlates of mTOR pathway activation in hepatocellular carcinoma and to determine whether rapamycin inhibits the pathway in cell culture. Experimental Design: Total and phosphorylated mTOR and S6K protein expression were studied by immunohistochemistry in hepatocellular carcinomas (n = 73), fibrolamellar carcinomas (n = 13), and hepatic adenomas (n = 15). Results were correlated with tumor growth pattern as defined by the WHO (trabecular, pseudoglandular/acinar, compact, and scirrhous), tumor size, Ki-67 proliferation index, and the modified Edmondson nuclear grade, which has a scale of 1 to 4. HepG2 and Hep3B cell lines were treated with rapamycin to see the effect on proliferation and S6K phosphorylation. Results: Increased expression of total mTOR was seen in 5% of hepatocellular carcinoma, whereas overexpression of phospho-mTOR was evident in 15% of hepatocellular carcinoma. Phospho-mTOR positivity correlated with increased expression of total S6K, which was found in 45% of cases. Total S6K overexpression was positively correlated with tumor nuclear grade, inversely with tumor size, and was unassociated with the proliferation index or WHO growth pattern. Rapamycin treatment of HepG2 and Hep3B cell lines markedly inhibited cell proliferation and reduced S6K phosphorylation in both cell lines. Conclusions: The mTOR pathway is activated in a subset of hepatocellular carcinoma. Rapamycin can inhibit proliferation of neoplastic hepatocytes in cell culture.

Immunohistochemical Labeling for Dpc4 Mirrors Genetic Status in Pancreatic Adenocarcinomas: A New Marker of DPC4 Inactivation

DPC4 (MADH4, SMAD4) is a tumor suppressor gene inactivated by allelic loss in approximately 55% of pancreatic adenocarcinomas. Unfortunately, it can be technically very difficult to detect the inactivation of DPC4 at the genetic level because genetic analyses require the microdissection of relatively pure samples of neoplastic and normal tissues. This is especially true for pancreatic adenocarcinomas, which elicit vigorous, non-neoplastic, stromal responses. Immunohistochemical labeling can overcome this hurdle because it preserves morphological information. We therefore studied the expression of the DPC4 gene product in 46 cancers, including 5 cancer cell lines by Western blot analysis and 41 primary periampullary adenocarcinomas by immunohistochemistry. The status of exons 1-11 of the DPC4 gene in all 46 of the cancers had been previously characterized at the molecular level, allowing us to correlate Dpc4 expression directly with gene status. Three cell lines had wild-type DPC4 genes, and Dpc4 expression was detected in all three by Western blot. The two cell lines with homozygously deleted DPC4 genes did not show Dpc4 protein by Western blot analysis. Immunohistochemical labeling revealed that 17 (94%) of the 18 primary adenocarcinomas with wild-type DPC4 genes expressed the DPC4 gene product, whereas 21 (91%) of 23 primary adenocarcinomas with inactivated DPC4 genes did not. Cases in which there was discordance between the immunohistochemical labeling and the genetic analyses were reanalyzed genetically, and we identified a deletion in exon 0 of DPC4 in one of these cases. This is the first report of a mutation in exon 0 of DPC4 in a pancreatic cancer. The contrast between the strong expression of Dpc4 by normal tissues and the loss of expression in the carcinomas was highlighted in several cases in which an infiltrating cancer was identified growing into a benign duct. These observations suggest that immunohistochemical labeling for the DPC4 gene product is an extremely sensitive and specific marker for DPC4 gene alterations in pancreatic carcinomas. The sensitivity and specificity of immunohistochemical labeling for Dpc4 in other periampullary carcinomas has yet to be determined.

SPARC/osteonectin is a frequent target for aberrant methylation in pancreatic adenocarcinoma and a mediator of tumor-stromal interactions.

Deregulated expression of SPARC/osteonectin, a secreted glycoprotein with multiple biological functions, has been associated with the progression of various cancers. Using microarrays, we previously identified SPARC as one of the genes induced by treatment with a DNA methylation inhibitor in pancreatic cancer cells. We therefore analysed the expression pattern and methylation status of the SPARC gene in pancreatic cancer. Gene expression profiling by oligonucleotide microarray and reverse transcription–PCR analyses demonstrated that SPARC mRNA was expressed in non-neoplastic pancreatic ductal epithelial cells, but was not expressed in a majority of pancreatic cancer cell lines. The loss of SPARC expression was associated with aberrant hypermethylation of its CpG island. Immunohistochemical labeling revealed that the SPARC protein was overexpressed in the stromal fibroblasts immediately adjacent to the neoplastic epithelium in primary pancreatic cancers, but rarely expressed in the cancers themselves. Primary fibroblasts derived from pancreatic cancer strongly expressed SPARC mRNA and secreted SPARC protein into the conditioned media, and treatment of pancreatic cancer cells with exogenous SPARC resulted in growth suppression. SPARC expression in fibroblasts from noncancerous pancreatic tissue was augmented by coculture with pancreatic cancer cells. These findings suggest that SPARC is a frequent target for aberrant methylation in pancreatic cancer and that SPARC expression in fibroblasts adjacent to pancreatic cancer cells is regulated through tumor–stromal interactions.

PIK3CA Mutations in Head and Neck Squamous Cell Carcinoma

Purpose: Recent studies have reported high frequencies of somatic mutations in the phosphoinositide-3-kinase catalytic α (PIK3CA) gene in several human solid tumors. Although gene amplifications of PIK3CA have been reported in head and neck squamous cell carcinoma (HNSCC), small mutation of the gene has not been evaluated in HNSCC previously. In this study, we examined the mutation frequency of PIK3CA in HNSCC. Experimental Design: More than 75% of the somatic mutations of PIK3CA are clustered in the helical (exon 9) and kinase domains (exon 20). To investigate the possible role of PIK3CA in HNSCC tumorigenesis, exons 1, 4, 5, 6, 7, 9, and 20 of the gene were analyzed by direct genomic DNA sequencing in 38 HNSCC specimens. Results: We identified four missense mutations in the seven exons of PIK3CA from 38 HNSCC specimens (11%). Three of the four mutations (i.e., H1047R, E542K, and E545K) have been previously reported as hotspot mutations. The remaining novel mutation, Y343C, is identified at exon 4 nucleotide 1028 A → G. Three of the four mutations were shown to be somatic, whereas the fourth mutation (H1047R) was identified in a cell line. Interestingly, three of the four mutations identified were in pharyngeal cancer samples. Conclusions: These data provide evidence that oncogenic properties of PIK3CA contribute to the carcinogenesis of human head and neck cancers, especially in pharyngeal cancer. A specific kinase inhibitor to PIK3CA may potentially be an effective therapeutic reagent against HNSCC or pharyngeal cancer in particular.

Defective B cell receptor‐mediated responses in mice lacking the Ets protein, Spi‐B

Spi‐B is a hematopoietic‐specific Ets family transcription factor closely related to PU.1. Previous gene targeting experiments have shown that PU.1 is essential for the production of both lymphocytes and monocytes. We have now generated mice with a null mutation at the Spi‐B locus. Unlike PU.1 mutant mice, Spi‐B−/− mice are viable, fertile and possess mature B and T lymphocytes. However, Spi‐B−/− mice exhibit severe abnormalities in B cell function and selective T cell‐dependent humoral immune responses. First, although Spi‐B−/− splenic B cells respond normally to lipopolysaccharide stimulation in vitro, these B cells proliferate poorly and die in response to B cell receptor (surface IgM) cross‐linking. Secondly, Spi‐B−/− mice display abnormal T‐dependent antigenic responses in vivo and produce low levels of antigen‐specific IgG1, IgG2a and IgG2b after immunization. Finally, Spi‐B−/− mice show a dramatic defect in germinal center formation and maintenance. In contrast to wild‐type animals, germinal centers in Spi‐B−/− mice are smaller and short‐lived with significantly increased numbers of apoptotic B cells. Taken together, these results demonstrate that Spi‐B is essential for antigen‐dependent expansion of B cells, T‐dependent immune responses and maturation of normal germinal centers in vivo.

PIK3CA Mutations in Intraductal Papillary Mucinous Neoplasm/Carcinoma of the Pancreas

Purpose: Recent studies have reported high frequencies of somatic mutations in the phosphoinositide-3-kinase catalytic-α (PIK3CA) gene in various human solid tumors. More than 75% of those somatic mutations are clustered in the helical (exon 9) and kinase domains (exon 20). The three hot-spot mutations, E542K, E545K, and H1047R, have been proven to elevate the lipid kinase activity of PIK3CA and activate the Akt signaling pathway. The mutational status of PIK3CA in intraductal papillary mucinous neoplasm/carcinoma (IPMN/IPMC) has not been evaluated previously. Experimental Design: To evaluate a possible role for PIK3CA in the tumorigenesis of IPMN and IPMC, exons 1, 4, 5, 6, 7, 9, 12, 18, and 20 were analyzed in 36 IPMN/IPMC and two mucinous cystadenoma specimens by direct genomic DNA sequencing. Results: We identified four missense mutations in the nine screened exons of PIK3CA from 36 IPMN/IPMC specimens (11%). One of the four mutations, H1047R, has been previously reported as a hot-spot mutation. The remaining three mutations, T324I, W551G, and S1015F, were novel and somatic. Conclusion: This is the first report of PIK3CA mutation in pancreatic cancer. Our data provide evidence that the oncogenic properties of PIK3CA contribute to the tumorigenesis of IPMN/IPMC.

Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms.

The suppressor of cytokine signalling-1 (SOCS-1) gene is frequently silenced in human hepatocellular carcinoma by aberrant methylation. The aim of this study was to determine if SOCS-1 is inactivated in pancreatic ductal neoplasms, and to investigate if aberrant methylation of this gene affected the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Aberrant methylation in the CpG island of the SOCS-1 gene was detected in six of 19 (31.6%) human pancreatic cancer cell lines using methylation-specific PCR, and was associated with a loss or reduction of gene expression in five of the six methylated cell lines. Thirteen of 60 pancreatic ductal adenocarcinomas (21.7%) and two of 34 intraductal papillary mucinous neoplasms (IPMNs) (5.9%) had methylated SOCS-1. In contrast, SOCS-1 methylation was not seen in pancreatic normal ductal epithelia (zero out of 15), in pancreatic intraepithelial neoplasia (PanINs) (zero out of 49) or in the IPMNs without infiltrating cancer (zero out of 20). 5-Aza-2′-deoxycytidine treatment of the SOCS-1-methylated pancreatic cancer cell lines led to restoration of SOCS-1 gene expression. Interleukin-6, which has been shown to act through the JAK/STAT pathway to increase cell growth, induced modest time and dose-dependent cell proliferation in a SOCS-1-methylated cell line (PL10, P=0.015) but not in two unmethylated cell lines. These results indicate that loss of SOCS-1 gene is associated with transcriptional silencing and may have growth-promoting effects, and that its methylation is a useful marker of pancreatic cancer.

ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma

DPC4 is known to mediate signals initiated by type β transforming growth factor (TGFβ) as well as by other TGFβ superfamily ligands such as activin and BMP (bone morphogenic proteins), but mutational surveys of such non-TGFβ receptors have been negative to date. Here we describe the gene structure and novel somatic mutations of the activin type I receptor, ACVR1B, in pancreatic cancer. ACVR1B has not been described previously as a mutated tumor-suppressor gene.

PU.1 and Spi-B Are Required for Normal B Cell Receptor–Mediated Signal Transduction

PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.

Loss of Stk11/Lkb1 expression in pancreatic and biliary neoplasms

We have documented previously somatic mutations of STK11/LKB11, the gene responsible for Peutz-Jeghers syndrome (PJS), in a small proportion of sporadic pancreatic adenocarcinomas, intraductal papillary mucinous neoplasms (IPMNs), and biliary adenocarcinomas. In this report, we characterize the expression of Stk11, the protein product of the STK11 gene, in a larger series of pancreatic and biliary neoplasms. First, the specificity of the Stk11 antibody was established in 23 neoplasms (22 IPMNs and 1 biliary adenocarcinoma) with known STK11 gene status. Complete absence of labeling was seen in the neoplastic cells of 3 of the 3 (100%) cases with previously documented biallelic inactivation of the STK11 gene, whereas 16 of the 20 (80%) IPMNs, presumably with at least one wild-type STK11 gene, retained Stk11 expression in the neoplastic cells. The marked decrease or absence of Stk11 expression in four neoplasms with wild-type STK11 suggests that additional mechanisms may account for the lack of Stk11 expression. Subsequently, to further evaluate Stk11 expression in pancreatic and biliary neoplasms, tissue microarrays (TMAs) were constructed from a series of nearly 100 ductal adenocarcinomas and biliary neoplasms. Stk11 expression was lost in 4 of the 56 (7%) pancreatic adenocarcinomas and 1 of the 38 (2.6%) biliary cancers by immunohistochemistry; the absence of labeling was confirmed by repeated immunohistochemical labeling of complete tissue sections for the same cases. Thus, Stk11 expression is abrogated in a small proportion of pancreatic and biliary neoplasms. The inactivation of Stk11 in 27% (6/22) of IPMNs versus 7% (4/56) of pancreatic adenocarcinomas suggests genetic disparities in the pathogenesis of these closely related neoplasms. Immunohistochemical analysis for Stk11 expression may be a valid surrogate for genetic analysis of STK11 gene mutations in cancers.