Inactivation of Smad4 accelerates Kras(G12D)-mediated pancreatic neoplasia

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of <5%. Genetic analysis of PDAC patient samples has shown that specific disease-associated mutations are correlated with histologically defined stages of neoplastic progression in the ductal epithelium. Activating mutations in KRAS are almost uniformly present in early-stage disease, with subsequent inactivating mutations in p16(INK4A), p53, and SMAD4 occurring in more advanced lesions. In this study, we have tested whether the loss of Smad4 would cooperate with an activating Kras(G12D) mutation to promote progression to PDAC using the Pdx1-Cre transgenic system to activate Kras(G12D) and delete Smad4 in all pancreatic lineages including the ductal epithelium. Analysis of double-mutant mice showed that loss of Smad4 significantly accelerated the progression of pancreatic intraepithelial neoplasias (mPanIN) and promoted a high incidence of intraductal papillary mucinous neoplasia and active fibrosis compared with Pdx1-Cre;Kras(G12D) or Pdx1-Cre;Smad4(lox/lox) mice. Occasionally, double-mutant mice progressed to locally invasive PDAC with little evidence of metastases by 6 months of age and without the detectable loss of p53 or p16(Ink4A) expression or function. The loss of Smad4 only seemed to promote disease progression in the presence of the activated Kras(G12D) allele because we observed no abnormal pathology within the pancreata of 23 Pdx1-Cre;Smad4(lox/lox) animals that were analyzed up to 8 months of age. This indicates that Smad4 is dispensable for normal pancreatic development but is critical for at least partial suppression of multiple Kras(G12D)-dependent disease-associated phenotypes.     

Kras(G12D) and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas

Oncogenic Kras initiates pancreatic tumorigenesis, while subsequent genetic events shape the resultant disease. We show here that concomitant expression of Kras(G12D) and haploinsufficiency of the Smad4/Dpc4 tumor suppressor gene engenders a distinct class of pancreatic tumors, mucinous cystic neoplasms (MCNs), which culminate in invasive ductal adenocarcinomas. Disease evolves along a progression scheme analogous to, but distinct from, the classical PanIN-to-ductal adenocarcinoma sequence, and also portends a markedly different prognosis. Progression of MCNs is accompanied by LOH of Dpc4 and mutation of either p53 or p16. Thus, these distinct phenotypic routes to invasive adenocarcinoma nevertheless share the same overall mutational spectra. Our findings suggest that the sequence, as well as the context, in which these critical mutations are acquired helps determine the ensuing pathology.     

Kras(G12D) and p53 mutation cause primary intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver with an increasing incidence and poor prognosis. Preclinical studies of the etiology and treatment of this disease are hampered by the relatively small number of available IHCC cell lines or genetically faithful animal models. Here we report the development of a genetically engineered mouse model of IHCC that incorporates two of the most common mutations in human IHCC, activating mutations of Kras (Kras(G12D)) and deletion of p53. Tissue-specific activation of Kras(G12D) alone resulted in the development of invasive IHCC with low penetrance and long latency. Latency was shortened by combining Kras(G12D) activation with heterozygous or homozygous deletion of p53 (mean survival of 56 weeks vs. 19 weeks, respectively), which also resulted in widespread local and distant metastasis. Serial analysis showed that the murine models closely recapitulated the multistage histopathologic progression of the human disease, including the development of stroma-rich tumors and the premalignant biliary lesions, intraductal papillary biliary neoplasms (IPBN), and Von Meyenburg complexes (VMC; also known as biliary hamartomas). These findings establish a new genetically and histopathologically faithful model of IHCC and lend experimental support to the hypothesis that IPBN and VMC are precursors to invasive cancers.     

Identification of tumorigenic cells in Kras(G12D)-induced lung adenocarcinoma

We established an inducible Kras(G12D)-driven lung adenocarcinoma in CCSP-rtTA/TetO-Cre/LSL-Kras(G12D) mice that enable pursuits of the cellular and molecular processes involved in Kras-induced tumorigenesis. To investigate the cellular origin of this cancer, we first report a strategy using fluorescence-activated cell sorting fractionation that could highly enrich bronchiolar Clara and alveolar type II cells, respectively. The EpCAM(+)MHCII(-) cells (bronchiolar origin) were more enriched with tumorigenic cells in generating secondary tumors than EpCAM(+)MHCII(+) cells (alveolar origin) in primary tumors that had been already initiated with oncogenic Kras activation. In addition, secondary tumors derived from EpCAM(+)MHCII(-) cells showed diversity of tumor locations compared with those derived from EpCAM(+)MHCII(+) cells. In the alveolar region, secondary tumors from EpCAM(+)MHCII(-) cells expressed not only bronchiolar epithelial marker, panCK, but also differentiation marker, proSPC, consistent with the notion that cancer-initiating cells display not only the abilities for self-renewal but also the features of differentiation to generate heterogeneous tumors with phenotypic diversity. Furthermore, high level of ERK1/2 activation and colony-forming ability as well as lack of Sprouty-2 expression were also observed in EpCAM(+)MHCII(-) cells. Therefore, these results suggest that bronchiolar Clara cells are the origin of cells and tumorigenesis for Kras(G12D)-induced neoplasia in the lungs.     

Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer

Inherited heterozygous BRCA2 mutations predispose carriers to tissue-specific cancers, but somatic deletion of the wild-type allele is considered essential for carcinogenesis. We find in a murine model of familial pancreatic cancer that germline heterozygosity for a pathogenic Brca2 truncation suffices to promote pancreatic ductal adenocarcinomas (PDACs) driven by Kras(G12D), irrespective of Trp53 status. Unexpectedly, tumor cells retain a functional Brca2 allele. Correspondingly, three out of four PDACs from patients inheriting BRCA2(999del5) did not exhibit loss-of-heterozygosity (LOH). Three tumors from these patients displaying LOH were acinar carcinomas, which also developed only in mice with biallelic Brca2 inactivation. We suggest a revised model for tumor suppression by BRCA2 with implications for the therapeutic strategy targeting BRCA2 mutant cancer cells.     

Molecular progression of promoter methylation in intraductal papillary mucinous neoplasms (IPMN) of the pancreas

To understand the role of gene promoter methylation in neoplastic evolution and progression, the methylation changes associated with 15 candidate tumor suppressor genes were studied throughout stages of tumor progression involving intraductal papillary mucinous neoplasms (IPMN) of the pancreas. Genomic DNA from 28 pancreatic IPMN tissue samples, categorized histologically as non-invasive intraductal IPMN (n = 3), IPMN with carcinoma in situ (n = 7), IPMN with microinvasion <1 mm (n = 4), and infiltrative IPMN with associated adenocarcinoma (n = 14), was modified by bisulfite treatment and analyzed with methylation-specific PCR (MSP). Promoter methylation of at least one tumor suppressor gene was present in 26/28 (92%) of the IPMNs. The cell cycle control genes, p16 and p73, were methylated frequently (>50%) in both non-invasive and invasive tumors. APC methylation was discovered in <10% of the non-invasive IPMNs versus 45% of the IPMNs associated with infiltrative adenocarcinoma, P = 0.040. Mismatch repair genes, hMLH1 and MGMT, were frequently methylated in the invasive IPMNs compared with the non-invasive tumors (38 versus 10% and 45 versus 20%, respectively) as was E-cadherin (38 versus 10%), P = 0.11. Multiple gene methylation at greater than three loci was present in 55% of the invasive tumors compared with 20% of the non-invasive tumors, P = 0.075. Lymph node status did not predict multi-gene methylation among tumors associated with invasive cancer. Compared with non-invasive IPMNs of the pancreas, IPMNs associated with adenocarcinoma demonstrate higher rates of aberrant tumor suppressor gene methylation. The sequential acquisition of hypermethylation at multiple gene promoter sites may explain tumor progression in IPMNs and other malignancies. Detection of methylation within selected genes may afford an accurate diagnostic molecular marker and predictor of neoplastic behavior.     

Delayed progression of pancreatic intraepithelial neoplasia in a conditional Kras(G12D) mouse model by a selective cyclooxygenase-2 inhibitor

Pancreatic ductal adenocarcinomas are thought to arise from noninvasive, intraductal precursor lesions called pancreatic intraepithelial neoplasias (PanIN). The study of PanINs holds great promise for the identification of early detection markers and effective cancer-preventing strategies. Cyclooxygenase-2 (COX-2) represents an intriguing target for therapeutic and preventive approaches in various human malignancies. The aim of the present study was to evaluate the efficacy of a selective COX-2 inhibitor to prevent the progression of PanINs in a conditional Kras(G12D) mouse model. Offspring of LSL-KRAS(G12D) x PDX-1-Cre intercrosses were randomly allocated to a diet supplemented with the selective COX-2 inhibitor nimesulide (400 ppm) or a control diet. After 10 months, animals were sacrificed. Successful recombination in the pancreas was evaluated by PCR. The pancreas of KRAS(G12D);PDX-1-Cre mice was analyzed for the presence of murine PanINs. Animals fed the COX-2 inhibitor had significantly fewer PanIN-2 and PanIN-3 lesions than control animals (P < 0.05). Ten percent of all pancreatic ducts in the nimesulide-fed animals showed PanIN-2 or PanIN-3 lesions, whereas 40% of the pancreatic ducts in the control animals had PanIN-2 or PanIN-3 lesions. Intrapancreatic prostaglandin E(2) levels were reduced in nimesulide-fed animals. Immunohistochemistry confirmed COX-2 expression in early and late PanINs. In summary, we found that the selective COX-2 inhibitor nimesulide delays the progression of pancreatic cancer precursor lesions in a preclinical animal model. These data highlight the importance of COX-2 in the development of pancreatic cancer. Inhibition of COX-2 may represent an intriguing strategy to prevent pancreatic cancer in high-risk patients.     

胰腺囊性肿瘤诊疗及进展

胰腺囊性肿瘤(PCNs)是少见肿瘤,发病原因尚不明确,不良生活习惯（吸烟、饮酒、重咖啡、高脂高蛋白饮食等）、慢性胰腺炎、环境污染因素及遗传因素等是潜在致病因素。PCNs分为浆液性囊性肿瘤（SCN）、黏液性囊性肿瘤（MCN）、胰腺导管内乳头状黏液肿瘤(IPMN)和实性假乳头状瘤（SPN）四种类型。发病症状常不典型,早期诊断难。PCNs具有典型影像特点,单个影像检查技术对PCNs的准确性和局限性不同,CT检查在胰腺病变中仍是最基本、最主要的检查方式。MRI对于小的囊性病灶比CT更有优势。超声内镜（EUS）充分结合了内镜和超声检查的优势,与CT、MRI检查相辅相成,同时还可进行细针穿刺取病理及囊液分析。尽管PCNs大部分为良性,但只要达到切除标准,均应推荐患者进行手术治疗,严格遵循PCNs诊治流程,制订个体化PCNs治疗策略,使患者利益最大化。     

胰腺黏液性囊性肿瘤的诊治进展

胰腺囊性肿瘤是一种比较少见的胰腺肿瘤,主要包括黏液性囊性肿瘤、浆液性囊性肿瘤、导管内乳头状黏液瘤。其中,胰腺黏液性囊性肿瘤是常见的原发性胰腺囊性肿瘤之一,由于它是一种具有潜在恶性的肿瘤,故术前诊断具有重要意义。近年来,随着现代影像技术如 CT、MRI 及 EUS 等的提高和完善,术前胰腺黏液性囊性肿瘤的诊断率比以往已有大大提高,但在治疗方面仍然没有统一的指南或规范。本文着重对胰腺黏液性囊性肿瘤的诊断、治疗进展做一综述。     

Potential pitfalls in the management of primary pancreatic cystic neoplasms

Primary pancreatic cystic neoplasms (PCN) have been increasingly recognized over the last 2 decades, mainly as a result of the widespread use of modern imaging techniques in patients with abdominal complaints. Biological behavior and management greatly differ between subgroups of PCN. Accurate preoperative diagnosis is required to select the optimal management strategy for each individual patient with PCN. An in-depth knowledge of the biological, imaging, macroscopic/microscopic features, and laboratory findings of each subtype of PCN is required on the part of the clinician, in order to select a cost-effective diagnostic evaluation of the patient. Clinical judgment is also required on the part of the surgeon to select the optimal procedure and to avoid severe pitfalls in the surgical management of these neoplasms.     

12-lipoxygenase metabolite 12(S)-HETE stimulates human pancreatic cancer cell proliferation via protein tyrosine phosphorylation and ERK activation.

We previously reported that inhibition of the 12-lipoxygenase pathway abolished proliferation and induced apoptosis in several pancreatic cancer cell lines. Furthermore, the 12-lipoxygenase product 12(S)-HETE stimulated pancreatic cancer cell proliferation and reversed 12-lipoxygenase inhibitor-induced growth inhibition. We investigated the underlying mechanism for 12(S)-HETE-induced pancreatic cancer cell proliferation, using 2 human pancreatic cancer cell lines, PANC-1 and HPAF. Cell proliferation was monitored by both thymidine incorporation and cell number. Western blotting was used to investigate the effect of 12(S)-HETE on cellular protein tyrosine phosphorylation as well as ERK, P38 MAPK and JNK/SAPK phosphorylation. 12(S)-HETE markedly stimulated proliferation of pancreatic cancer cells in a time- and concentration-dependent manner. In parallel, 12(S)-HETE induced tyrosine phosphorylation of multiple cellular proteins, while inhibition of tyrosine kinase by genestein abolished 12(S)-HETE-induced proliferation, indicating that intracellular protein tyrosine kinase activation is involved in the mitogenic effects of 12(S)-HETE. Following treatment with 12(S)-HETE, both ERK and P38 MAPK, but not JNK/SAPK, were phosphorylated. The specific MEK inhibitors PD098059 and U0126, which in turn suppress ERK, abolished 12(S)-HETE-stimulated proliferation. In contrast, inhibition of P38 MAPK with SB203580 did not affect 12(S)-HETE-stimulated pancreatic cancer cell proliferation. Furthermore, 12(S)-HETE-stimulated ERK phosphorylation was inhibited by genestein, indicating that tyrosine phosphorylation is essential for ERK activation. These findings suggest that both ERK and cellular protein tyrosine kinase activation are involved in 12(S)-HETE-induced pancreatic cancer cell proliferation but P38 and JNK/SAPK are not involved in this mitogenic effect.     

Dual effect of Kras(D12) knockdown on tumorigenesis: increased immune-mediated tumor clearance and abrogation of tumor malignancy

Activating mutations in the human KRAS proto-oncogene are acquired during the earliest stages of colorectal cancer development. If mutant KRAS is to be used as a target for therapy in colorectal cancer, tumor growth should depend on its continued presence. Here, we report that stable knockdown of Kras(D12) in murine C26 colorectal cancer cells by RNA interference resulted in loss of transformed properties in vitro. The incidence of subcutaneous tumor formation was reduced by 60% and the lag time was increased sevenfold. Kras(D12)-knockdown tumors grew noninvasively and did not cause morbidity. Remarkably, some of the Kras(D12)-knockdown tumors regressed spontaneously, which rendered these mice resistant to parental C26 tumor growth. In immune-deficient hosts, the incidence of tumor formation by Kras(D12)-knockdown cells was 100%. None of these tumors regressed spontaneously. We conclude that the reduced incidence of tumor formation by Kras(D12)-knockdown cells is due to tumor cell clearance by the host immune system, but not to an intrinsic inability of these cells to grow out as tumors. Interestingly, Kras(D12) knockdown resulted in increased production of interleukin 18 (Il-18), an immune-stimulatory cytokine that has been implicated in limiting colorectal tumor formation. Thus, mutant Kras(D12) suppresses Il-18 production in colorectal tumor cells, which may contribute to evasion of the local immune system during tumor development.     

BRAF and KRAS gene mutations in intraductal papillary mucinous neoplasm/carcinoma (IPMN/IPMC) of the pancreas

The Raf/MEK/ERK (MAPK) signal transduction is an important mediator of a number of cellular fates including growth, proliferation, and survival. The BRAF gene is activated by oncogenic RAS, leading to cooperative effects in cells responding to growth factor signals. Our study was performed to elucidate a possible role of BRAF in the development of IPMN (Intraductal Papillary Mucinous Neoplasm) and IPMC (Intraductal Papillary Mucinous Carcinoma) of the pancreas. Mutations of BRAF and KRAS were evaluated in 36 IPMN/IPMC samples and two mucinous cystadenomas by direct genomic sequencing. Exons 1 for KRAS, and 5, 11, and 15 for BRAF were examined. Totally we identified 17 (47%) KRAS mutations in exon 1, codon 12 and one missense mutation (2.7%) within exon 15 of BRAF. The mutations appear to be somatic since the same alterations were not detected in the corresponding normal tissues. Our data provide evidence that oncogenic properties of BRAF contribute to the tumorigenesis of IPMN/IPMC, but at a lower frequency than KRAS.     

Rottlerin suppresses growth of human pancreatic tumors in nude mice,and pancreatic cancer cells isolated from Kras mice

The purpose of the study was to examine the molecular mechanisms by which rottlerin inhibited growth of human pancreatic tumors in Balb C nude mice, and pancreatic cancer cells isolated from Kras^G12D mice. AsPC-1 cells were injected subcutaneously into Balb c nude mice, and tumor-bearing mice were treated with rottlerin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of components of Akt, Notch, and Sonic Hedgehog (Shh) pathways were measured by the immunohistochemistry, Western blot analysis, and/or q-RT-PCR. The effects of rottlerin on pancreatic cancer cells isolated from Kras^G12D mice were also examined. Rottlerin-treated mice showed a significant inhibition in tumor growth which was associated with suppression of cell proliferation, activation of capase-3 and cleavage of PARP. Rottlerin inhibited the expression of Bcl-2, cyclin D1, CDK2 and CDK6, and induced the expression of Bax in tumor tissues compared to untreated control. Rottlerin inhibited the markers of angiogenesis (Cox-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thus blocking production of tumorigenic mediators in tumor microenvironment. Rottlerin also inhibited epithelial–mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Slug and Snail. Furthermore, rottlerin treatment of xenografted tumors or pancreatic cancer cells isolated from Kras^G12D mice showed a significant inhibition in Akt, Shh and Notch pathways compared to control groups. These data suggest that rottlerin can inhibit pancreatic cancer growth by suppressing multiple signaling pathways which are constitutively active in pancreatic cancer. Taken together, our data show that the rottlerin induces apoptosis and inhibits pancreatic cancer growth by targeting Akt, Notch and Shh signaling pathways, and provide a new therapeutic approach with translational potential for humans.     

A comparison between intraductal papillary neoplasms of the biliary tract (BT-IPMNs) and intraductal papillary mucinous neoplasms of the pancreas (P-IPMNs) reveals distinct clinical manifestations and outcomes

Background

Intraductal papillary mucinous neoplasm of the biliary tract (BT-IPMN) has been increasingly recognized as a biliary counterpart of intraductal papillary mucinous neoplasm of the pancreas (P-IPMN). However, there is limited information regarding whether BT-IPMNs and P-IPMNs behave in a similar fashion.

Methods

We retrospectively compared clinicopathological variables between 9 patients with BT-IPMN and 44 patients with P-IPMN.

Results

There was no significant difference in age between patients with BT-IPMN and those with P-IPMN. The male/female ratio was significantly higher in patients with P-IPMN than in those with BT-IPMN (P = 0.012). Clinical presentation with jaundice was more common in patients with BT-IPMN (67%) than in those with P-IPMN (4.5%, P = 0.002). In addition, serum levels of CEA and CA19-9 were higher in patients with BT-IPMN than in those with P-IPMN (P = 0.019 and P = 0.002, respectively). The pathological diagnosis of malignancy was significantly more common in patients with BT-IPMN (89%) than in those with P-IPMN (23%, P = 0.002). The association with invasive carcinoma was significantly more frequent in patients with BT-IPMN (44.4%) than in those with P-IPMN (6.8%, P = 0.008). Furthermore, survival time after surgical resection was significantly shorter in patients with BT-IPMN than in those with P-IPMN (P = 0.002).

Conclusion

These findings reveal differences in clinicopathological features and prognosis between BT-IPMN and P-IPMN, thereby suggesting distinct biological pathways underlying the pathogenesis of these neoplasms.     

胰腺囊性肿瘤222例临床诊治分析

目的 总结本中心收治的胰腺囊性肿瘤（PCN）的诊治情况及预后,为PCN的临床处理提供一定依据。方法 回顾性分析2005年1月至2016年10月南京鼓楼医院收治的PCN病例,分析各类PCN的临床特征、超声内镜下表现、治疗方式及预后情况。结果 本研究共纳入222例PCN病例,包括94例导管内乳头状黏液瘤（IPMN）,58例黏液性囊性肿瘤（MCN）,43例浆液性囊性肿瘤（SCN）及27例实性假乳头状肿瘤（SPN）。各型PCN患者的男女比例1∶1.55,平均年龄为（56.0±15.7）岁,大部分PCN患者（64.0％,142／222）在就诊时有临床表现,其中以腹痛最常见（47.3％,105／222）。各型PCN术后病理提示分别有7例IPMN（16.3％,7／43）和5例MCN（9.1％,5／55）为恶性（伴浸润性癌或重度异型增生）,而SCN和SPN术后病理均为良性（P=0.027）。未手术的PCN患者中,分别有4例IPMN（8.3％,4／48）和2例SPN（66.7％,2／3）在随访过程中发生癌变。获随访的188例PCN患者的5年生存率为86.0％。124例PCN患者术后的5年生存率为90.0%,SCN、MCN和SPN的术后5年生存率分别为100.0％、92.1％和938％,而IPMN仅为74.0％,差异有统计学意义（P=0.003）。结论 PCN好发于中年女性,大部分患者就诊时有临床症状,最常表现为腹痛。MCN、IPMN和SPN具有恶变倾向,应在符合手术条件的情况下行手术切除;而SCN则可采取保守治疗。各类PCN预后差异显著,IPMN预后最差,MCN、SCN及SPN术后预后均较好。