Eric K. Nakakura

MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer

While altered expression of microRNAs (miRs) in tumors has been well documented, it remains unclear how the miR transcriptome intersects neoplastic progression. By profiling the miR transcriptome we identified miR expression signatures associated with steps in tumorigenesis and the acquisition of hallmark capabilities in a prototypical mouse model of cancer. Metastases and a rare subset of primary tumors shared a distinct miR signature, implicating a discrete lineage for metastatic tumors. The miR-200 family is strongly down-regulated in metastases and met-like primary tumors, thereby relieving repression of the mesenchymal transcription factor Zeb1, which in turn suppresses E-cadherin. Treatment with a clinically approved angiogenesis inhibitor normalized angiogenic signature miRs in primary tumors, while altering expression of metastatic signature miRs similarly to liver metastases, suggesting their involvement in adaptive resistance to anti-angiogenic therapy via enhanced metastasis. Many of the miR changes associated with specific stages and hallmark capabilities in the mouse model are similarly altered in human tumors, including cognate pancreatic neuroendocrine tumors, implying a generality.

Notch Signaling Induces Rapid Degradation of Achaete-Scute Homolog 1

ABSTRACT In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes which leads to transcriptional repression of neural determination and differentiation genes. We now report that in addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, we showed that the initial appearance of Notch1 coincided with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone was capable of down-regulating hASH1 mRNA but could not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 was coinfected with Notch1, we still observed a dramatic and abrupt loss of the exogenous hASH1 protein, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreased the apparent half-life of the adenoviral hASH1 protein and increased the fraction of hASH1 which was polyubiquitinylated. The proteasome inhibitor MG132 reversed the Notch1-induced degradation. The Notch RAM domain was dispensable but a lack of the OPA and PEST domains inactivated this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 could protect hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer.

Cyclin-Dependent Kinase 5 Activity Controls Cell Motility and Metastatic Potential of Prostate Cancer Cells

We show here that cyclin-dependent kinase 5 (CDK5), a known regulator of migration in neuronal development, plays an important role in prostate cancer motility and metastasis. P35, an activator of CDK5 that is indicative of its activity, is expressed in a panel of human and rat prostate cancer cell lines, and is also expressed in 87.5% of the human metastatic prostate cancers we examined. Blocking of CDK5 activity with a dominant-negative CDK5 construct, small interfering RNA, or roscovitine resulted in changes in the microtubule cytoskeleton, loss of cellular polarity, and loss of motility. Expression of a dominant-negative CDK5 in the highly metastatic Dunning AT6.3 prostate cancer cell line also greatly impaired invasive capacity. CDK5 activity was important for spontaneous metastasis in vivo; xenografts of AT6.3 cells expressing dominant-negative CDK5 had less than one-fourth the number of lung metastases exhibited by AT6.3 cells expressing the empty vector. These results show that CDK5 activity controls cell motility and metastatic potential in prostate cancer.

Pancreatic adenocarcinoma, version 2.2017: Clinical practice guidelines in Oncology

Ductal adenocarcinoma and its variants account for most pancreatic malignancies. High-quality multiphase imaging can help to preoperatively distinguish between patients eligible for resection with curative intent and those with unresectable disease. Systemic therapy is used in the neoadjuvant or adjuvant pancreatic cancer setting, as well as in the management of locally advanced unresectable and metastatic disease. Clinical trials are critical for making progress in treatment of pancreatic cancer. The NCCN Guidelines for Pancreatic Adenocarcinoma focus on diagnosis and treatment with systemic therapy, radiation therapy, and surgical resection.

Mammalian Scratch: A neural-specific Snail family transcriptional repressor

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix–loop–helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

Lymph node sampling rates and predictors of nodal metastasis in pancreatic neuroendocrine tumor resections: the UCSF experience with 149 patients.

Objectives The decision to perform pancreas-preserving procedures or standard resections for pancreatic neuroendocrine tumors (PNETs) is often based on the perceived risk of malignancy, including potential nodal involvement. We sought to identify clinicopathological factors that predict nodal disease. Methods This is a retrospective review of pathology database for PNET resections from January 1, 1988, to March 15, 2010. Univariate analysis and multivariate logistic regression were used to identify predictors of nodal metastasis. Results A total of 149 patients were identified. Enucleations had lower lymph node sampling rates compared to major resections. Excluding enucleations, 23% of patients had no lymph nodes sampled. For patients who did have lymph nodes evaluated, a median of 5 lymph nodes were examined. On multivariate analysis, only distant disease predicted nodal metastasis (odds ratio = 3.80, P = 0.02); tumor size did not (P = 0.48). One third of patients with lymph node metastasis had tumors less than 3 cm. Conclusions Lymph nodes are not evaluated in many major pancreatic resections for PNET, and preoperative prediction of nodal metastasis is difficult, even when tumor size is considered. Consequently, many patients may be understaged and undergo potentially inadequate resection. Inconsistent lymph node sampling may explain conflicting conclusions in the literature regarding the prognostic value of lymph node involvement in PNET patients.

Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

Unique patterns of metastases in common and rare types of malignancy.

This review on the unique patterns of metastases by common and rare types of cancer addresses regional lymphatic metastases but also demonstrates general principles by consideration of vital organ metastases. These general features of successfully treated metastases are relationships to basic biological behavior as illustrated by disease‐free interval, organ‐specific behavior, oligo‐metastatic presentation, genetic control of the metastatic pattern, careful selection of patients for surgical resection, and the necessity of complete resection of the few patients eligible for long‐term survival after resection of vital organ metastasis. Lymph node metastases, while illustrating these general features, are not related to overall survival because lymph node metastases themselves do not destroy a vital organ function, and therefore have no causal relationship to overall survival. When a cancer cell spreads to a regional lymph node, does it also simultaneously spread to the systemic site or sites? Alternatively, does the cancer spread to the regional lymph node first and then it subsequently spreads to the distant site(s) after an incubation period of growth in the lymph node? Of course, if the cancer is in its incubation stage in the lymph node, then removal of the lymph node in the majority of cases with cancer cells may be curative. The data from the sentinel lymph node era, particularly in melanoma and breast cancer, is consistent with the spectrum theory of cancer progression to the sentinel lymph node in the majority of cases prior to distant metastasis. Perhaps, different subsets of cancer may be better defined with relevant biomarkers so that mechanisms of metastasis can be more accurately defined on a molecular and genomic level. J. Surg. Oncol. 2011;103:607–614.

Homeodomain transcription factor NKX2.2 functions in immature cells to control enteroendocrine differentiation and is expressed in gastrointestinal neuroendocrine tumors

The homeodomain transcription factor NKX2.2 is necessary for neuroendocrine (NE) differentiation in the central nervous system and pancreas. NE tumors derived from the gut are defined by their NE phenotype, which is used for diagnosis and contributes to tumorigenicity. We hypothesized that NKX2.2 is important for NE differentiation in normal and neoplastic gut. NKX2.2 and NE marker expression was investigated in the small intestine of embryonic and adult mice using immunofluorescence (IF). To determine the role of NKX2.2 in NE differentiation of the intestine, the phenotype of Nkx2.2 (-/-) mice was examined by IF and real-time (RT)-PCR. NKX2.2 and NE marker expression in human NE tumors of the gut and normal tissues were evaluated by immunohistochemistry and qRT-PCR. NKX2.2 expression was detected in the intervillus/crypt regions of embryonic and adult mouse intestine. Co-expression of Nkx2.2 with neurogenin3 (NEUROG3) and hormones was observed in the adult intestinal crypt compartment, suggesting NKX2.2 functions in NEUROG3-positive endocrine progenitors and newly differentiated endocrine cells. In the intestine of Nkx2.2 (-/-) mice, we found a dramatic reduction in the number of cells producing numerous hormones, such as serotonin, gastrin, cholecystokinin, somatostatin, glucagon-like peptide 1 (GLP-1), and secretin, but an increase in cells producing ghrelin. NKX2.2 was expressed in most (24 of 29) human NE tumors derived from diverse primary sites. We conclude NKX2.2 functions in immature endocrine cells to control NE differentiation in normal intestine and is expressed in most NE tumors of the gut, and is therefore a novel target of diagnosis for patients with gastrointestinal NE tumors.

Pseudomyxoma Peritonei: More Questions Than Answers

Chances are, if you ask most physicians and surgeons about pseudomyxoma peritonei (PMP), they will respond with more questions than answers. The confusion that surrounds PMP is not surprising because the origin, pathology, treatment, prognosis, and very definition of PMP are still under debate. PMP is a clinical syndrome that is characterized by mucinous ascites that result from rupture of a mucin-producing neoplasm, typically of appendiceal origin. Whereas surgical resection alone of an intact, mucin-producing neoplasm of the appendix (mucocele) is curative, rupture of an appendiceal mucocele may lead to diffuse peritoneal spread of mucin that may contain epithelium of a low-grade or high-grade nature (ie, PMP), which causes fatal bowel obstruction without treatment. The appendix is the primary cause of PMP; the ovaries are typically only secondarily involved. The two types of mucin-producing appendiceal neoplasms that give rise to PMP exhibit a different biology and prognosis. The outcome is consistently more favorable for patients with PMP that results from the rupture of a mucin-producing adenoma of the appendix (low-grade process) than from the rupture of a mucin-producing appendiceal adenocarcinoma (high-grade process). A detailed pathologic classification of PMP is therefore critical to define patient prognosis, to determine the appropriate treatment, and to permit comparison of results among studies. Unfortunately, the pathologic classification of PMP is controversial and confusing. In 1995, Ronnett et al described three distinct categories of PMP on the basis of pathologic specimens (n 109): diffuse peritoneal adenomucinosis (DPAM), peritoneal mucinous carcinoma (PMCA), and an intermediate/discordant subtype (PMCA-I/D). DPAM originates from an appendiceal mucinous adenoma and produces abundant mucin but minimal mucinous epithelium that lacks significant cytologic atypia and mitoses. The prognosis for DPAM is good. An appendiceal adenocarcinoma causes PMCA, which features more mucinous epithelium and pathology that is typical of adenocarcinoma. The prognosis for PMCA is poor. The third category, PMCA-I/D, has characteristics of both DPAM and PMCA, and the prognosis is intermediate between the two. When the same surgeon treated patients with PMP tumors uniformly, the 5-year overall survival was 75% for DPAM, 50% for PMCA-I/D, and 14% for PMCA. This range suggests inherent differences in biology among DPAM, PMCA, and PMCA-I/D tumors that greatly affect patient prognosis. Two different two-group classifications have been proposed. One, by Bradley et al, is based on an analysis of patients with PMP of appendiceal origin (n 101). It consists of low-grade mucinous carcinoma peritonei, which includes DPAM and PMCA-I/D tumors, and high-grade mucinous carcinoma peritonei, which includes PMCA. When patients were treated uniformly, the 5-year overall survival did not differ significantly (68% for DPAM and 62% for PMCA-I/D). The second two-group classification of PMP was proposed by Misdraji et al and was also based on an analysis of patients with PMP of appendiceal origin (n 52). It also consists of low-grade and high-grade tumors. All of these classifications, based as they are on a limited number of patients, leave it unclear whether a two-or threegroup classification system best categorizes patients with PMP into unique prognostic groups. In the report that accompanies this editorial, Chua et al examine the outcome of nearly 2,300 patients from 16 institutions worldwide who were treated uniformly over an 18-year period for PMP that arose from the appendix. A major strength of this registry study is the detailed pathologic classification of PMP as either DPAM, PMCA, or PMCA-I/D (hybrid) tumors. The study confirmed the paramount importance of PMP histopathologic subtype with respect to outcome. Five-year overall survival was 81% for DPAM, 78% for hybrid tumors, and 59% for PMCA. Multivariable analysis showed that the PMCA histopathologic subtype was an independent predictor of poorer overall survival. Interestingly, the outcome seemed similar for DPAM and for hybrid tumors, supporting a two-group, low-grade versus highgrade classification system, as previously proposed by Bradley et al. Future classification of patients with PMP of appendiceal origin into low-grade (DPAM, hybrid tumors) and high-grade (PMCA) groups should permit the meaningful comparison of results among studies. Aside from underlying tumor biology, another important, previously described prognostic factor for patients with PMP is the ability to remove all gross disease; this concept was supported by the study by Chua et al. Intuitively, this makes sense because the cause of death for most patients with PMP is multifocal bowel obstruction. Five-year overall survival was 85% when no macroscopic residual cancer remained (completeness of cytoreduction [CCR] score of 0) and 80% when no nodule greater than 2.5 mm in diameter remained (CCR1). In contrast, 5-year overall survival was only 24% when gross residual tumor remained (CCR2/3), which was significantly worse. Importantly, most patients with PMP in this study had a CCR0 (51%) or a CCR1 (32%) score, and only a few (17%) underwent debulking surgery (CCR2/3). Does the high proportion of patients with CCR0 and JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 30 NUMBER 20 JULY 1