Gina M. DeNicola

Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a Mouse Model of Pancreatic Cancer

Its All in the Delivery Pancreatic cancer is almost universally associated with a poor prognosis, in part because the tumors are resistant to chemotherapeutic drugs. Working with a mouse tumor model that displays many features of the human disease, Olive et al. (p. 1457, published online 21 May; see the Perspective by Olson and Hanahan) found that the tumors were poorly vascularized, a factor likely to impede drug delivery. Treatment of the mice with the chemotherapeutic drug gemcitabine in combination with a drug that depletes tumor-associated stromal tissue led to an increase in tumor vasculature, enhanced delivery of gemcitabine, and a delay in disease progression. Thus, drugs targeting the tumor stroma may merit investigation as a way to enhance the efficacy of conventional chemotherapy for pancreatic cancer. Pancreatic tumors are unresponsive to chemotherapy because their limited vasculature precludes efficient drug delivery. Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.

Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis

Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 (also known as Nfe2l2) and its repressor protein Keap1 (refs 2–5). In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2–Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, indicating that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of Kras, Braf and Myc, and found that ROS are actively suppressed by these oncogenes. K-RasG12D, B-RafV619E and MycERT2 each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a new mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-RasG12D and B-RafV619E, and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-RasG12D-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin‐dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone‐dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

NRF2 regulates serine biosynthesis in non–small cell lung cancer

Tumors have high energetic and anabolic needs for rapid cell growth and proliferation, and the serine biosynthetic pathway was recently identified as an important source of metabolic intermediates for these processes. We integrated metabolic tracing and transcriptional profiling of a large panel of non–small cell lung cancer (NSCLC) cell lines to characterize the activity and regulation of the serine/glycine biosynthetic pathway in NSCLC. Here we show that the activity of this pathway is highly heterogeneous and is regulated by NRF2, a transcription factor frequently deregulated in NSCLC. We found that NRF2 controls the expression of the key serine/glycine biosynthesis enzyme genes PHGDH, PSAT1 and SHMT2 via ATF4 to support glutathione and nucleotide production. Moreover, we show that expression of these genes confers poor prognosis in human NSCLC. Thus, a substantial fraction of human NSCLCs activates an NRF2-dependent transcriptional program that regulates serine and glycine metabolism and is linked to clinical aggressiveness.

C-Raf is required for the initiation of lung cancer by K-Ras(G12D).

The Ras/Raf/MEK/ERK (extracellular signal-regulated kinase) pathway is primarily responsible for mitogenesis in metazoans, and mutational activation of this pathway is common in cancer. A variety of selective chemical inhibitors directed against the mitogen-activated protein kinase pathway are now available for clinical investigation and thus the determination of the importance of each of the kinases in oncogenesis is paramount. We investigated the role of two Raf kinases, B-Raf and C-Raf, in Ras oncogenesis, and found that although B-Raf and C-Raf have overlapping functions in primary mesenchymal cells, C-Raf but not B-Raf is required for the proliferative effects of K-Ras(G12D) in primary epithelial cells. Furthermore, in a lung cancer mouse model, C-Raf is essential for tumor initiation by oncogenic K-Ras(G12D), whereas B-Raf is dispensable for this process. Our findings reveal that K-Ras(G12D) elicits its oncogenic effects primarily through C-Raf and suggest that selective C-Raf inhibition could be explored as a therapeutic strategy for K-Ras-dependent cancers.

C-Raf Inhibits MAPK Activation and Transformation by B-RafV600E

Activating B-Raf mutations that deregulate the MAPK pathway commonly occur in cancer. Whether additional proteins modulate the enzymatic activity of oncogenic B-Raf is unknown. Here we show that the proto-oncogene C-Raf paradoxically inhibits B-Raf(V600E) kinase activity through the formation of B-Raf(V600E)-C-Raf complexes. Although all Raf family members associate with oncogenic B-Raf, this inhibitory effect is specific to C-Raf. Indeed, a B-Raf(V600E) isoform with impaired ability to interact with C-Raf exhibits elevated oncogenic potential. Human melanoma cells expressing B-Raf(V600E) display a reduced C-Raf:B-Raf ratio, and further suppression of C-Raf increases MAPK activation and proliferation. Conversely, ectopic C-Raf expression lowers ERK phosphorylation and proliferation. Moreover, both oncogenic Ras and Sorafenib stabilize B-Raf(V600E)-C-Raf complexes, thereby impairing MAPK activation. This inhibitory function of C-Raf on B-Raf(V600E)-mediated MAPK activation may explain the lack of co-occurrence of B-Raf(V600E) and oncogenic Ras mutations, and influence the successful clinical development of small molecule inhibitors for B-Raf(V600E)-driven cancers.

RAS in cellular transformation and senescence.

In 1964, a transforming retrovirus was described that produced tumours in mice [1]. The Harvey rat sarcoma virus, aptly named H-RAS after the discovering scientist, encoded an oncogene that had been hijacked from its host. A similar virus was isolated in 1970 and was named the Kirsten rat sarcoma virus or K-RAS [2]. In 1982, the human genes homologous to the viral genes were elucidated and were designated c-H-RAS and c-K-RAS [3], and subsequently the third and final RAS family member was isolated from human neuroblastoma samples and termed c-N-RAS. An examination of other human cancers also revealed RAS genes capable of transforming mammalian cells, and interestingly, these alleles were transforming due to point mutations that inactivated the proteins’ intrinsic guanine triphosphatase (GTPase) activity [4,5]. RAS functions downstream of mitogenic growth factor receptors. Following the ligation with growth factors, these receptor tyrosine kinases undergo autophosphorylation and thereby recruit adaptor proteins that bind guanine nucleotide exchange factors (GEFs) (for review, see [6]). GEFs catalyse the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) on small GTP-binding proteins, including RAS. GTP-bound RAS subsequently interacts with a number of effectors to regulate cellular proliferation and survival, notably including the RAF family of proteins and type I phosphoinositol-3-kinase (PI3K). RAS proteins mutated at codons 12, 13, or 61 are rendered constitutively GTP bound and consequently have increased affinity to RAF proteins and PI3K, leading to activation of downstream pathways. The RAF family of serine/threonine kinases consists of three members: ARAF, BRAF, and CRAF [6]. Following activation by RAS, RAF kinases phosphorylate Mitogen-Activated Protein Kinase (MEK) kinases (MEK1 and MEK2), resulting in their activation. MEK proteins subsequently phosphorylate Extracellular Regulated MAP Kinase (ERK) (ERK1 and ERK2) in the cytoplasm, resulting in their activation and nuclear translocation. ERK kinases have a variety of cytoplasmic and nuclear targets, importantly including ETS transcription factors to regulate the expression of many pro-proliferative genes. RAS-GTP also interacts with and stimulates the activity of PI3K [6]. Upon activation, PI3K catalyses the conversion of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to produce phosphatidylinositol-3,4,5trisphosphate (PtdIns(3,4,5)P3). Increased local concentrations of PtdIns(3,4,5)P3 result in recruitment of the kinase PDK1 via its pleckstrin homology domain, which phosphorylates and activates the kinase AKT. AKT in turn promotes cellular survival and activates mammalian target of rapamycin (mTOR) to increase ribosomal biogenesis and messenger RNA (mRNA) translation. Additionally, active PI3K also stimulates the GTP-binding protein RAC, a regulator of the actin cytoskeleton. Therefore, rather than relying upon a single pathway for cellular transformation, it is hypothesised that numerous alterations in multiple biochemical pathways collectively promote cellular transformation by oncogenic RAS. Oncogenic RAS mutations are the most common oncogenic events identified in human tumours, being found in 30% of human cancers. KRAS is the most commonly mutated member of the RAS family, present in over 90% of ductal pancreatic cancers, 40−50% colorectal cancers, and 30% of non-small cell lung cancers (6). A detailed characterisation of the cellular and molecular pathways altered by oncogenic RAS has been pursued in order to identify potential therapeutic targets for such cancers, but despite its expected role in promoting tumourigenesis, ectopic expression of oncogenic RAS in primary cell cultures induces a paradoxical irreversible growth arrest known as oncogene-induced senescence (OIS) [7]. OIS is characterised by a flattened cellular morphology, a large nucleus with a prominent nucleolus, chromatin reorganisation, and activation of the p53 and p16INK4a pathways [7]. Although ectopic expression of oncogenic RAS in primary cells induces OIS, the exact molecular mechanisms remain unclear. Studies have

Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers

Significance Serine supports a number of anabolic processes, including protein, lipid, and nucleic acid synthesis. Cells can either import serine or synthesize it de novo. Recently, overexpression of 3-phosphoglycerate dehydrogenase (PHGDH), the gene encoding the first committed step of serine synthesis, via focal amplification and other mechanisms, has been identified in human cancers. Cancer cell lines that overexpress PHGDH are uniquely sensitive to PHGDH knockdown whereas lines that express little PHGDH are insensitive, suggesting that PHGDH may be a clinically interesting target. Here, we report the discovery of a specific small molecule inhibitor of PHGDH, which enables preclinical evaluation of PHGDH as a target in cancer and provides a tool to study the biology of de novo serine synthesis. Cancer cells reprogram their metabolism to promote growth and proliferation. The genetic evidence pointing to the importance of the amino acid serine in tumorigenesis is striking. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of serine biosynthesis, is overexpressed in tumors and cancer cell lines via focal amplification and nuclear factor erythroid-2-related factor 2 (NRF2)-mediated up-regulation. PHGDH-overexpressing cells are exquisitely sensitive to genetic ablation of the pathway. Here, we report the discovery of a selective small molecule inhibitor of PHGDH, CBR-5884, identified by screening a library of 800,000 drug-like compounds. CBR-5884 inhibited de novo serine synthesis in cancer cells and was selectively toxic to cancer cell lines with high serine biosynthetic activity. Biochemical characterization of the inhibitor revealed that it was a noncompetitive inhibitor that showed a time-dependent onset of inhibition and disrupted the oligomerization state of PHGDH. The identification of a small molecule inhibitor of PHGDH not only enables thorough preclinical evaluation of PHGDH as a target in cancers, but also provides a tool with which to study serine metabolism.

Cathepsin B promotes the progression of pancreatic ductal adenocarcinoma in mice

Objective The lysosomal protease cathepsin B is upregulated in human pancreatic ductal adenocarcinoma (PDA) and represents a potential therapeutic target. Loss of cathepsin B delays tumour progression in mouse models of islet, mammary and intestinal carcinoma and decreases invasion and metastasis. This study examines the role of cathepsin B in the initiation, progression and metastasis of PDA. Methods Cathepsin B germline knockout mice were crossed with animals expressing an endogenous KrasG12D allele in the pancreas, and mice were aged to evaluate the role of cathepsin B in pancreatic intraepithelial neoplasia (PanIN). A survival study was also performed with mice carrying an additional heterozygous conditional Trp53R172H allele. Cell lines derived from tumours were used to investigate the role of cathepsin B in vitro, and subcutaneous allografts investigated the cell autonomous and non-cell autonomous roles of cathepsin B in pancreatic cancer. Results Constitutive cathepsin B loss resulted in delayed progression of both PanIN and PDA and a significant survival advantage in mice. Cathepsin B-deficient PDA cells and PanIN showed decreased proliferation and mitogen-activated protein (MAP) kinase signalling. The reconstitution of deficient cells with cathepsin B reversed these findings, which correlated with decreased levels of the active forms of the related protease cathepsin L. Conversely, acute ablation of cathepsin L activated the MAP kinase cascade in PDA cells. Conclusions These results confirm that cathepsin B plays an important cell autonomous role in the progression of PDA and suggest that the regulation of cathepsin L by cathepsin B may be a means of stimulating cell proliferation in neoplasia.

VAV1: A new target in pancreatic cancer?

Pancreatic ductal adenocarcinoma (PDA) is arguably the most lethal malignancy in the United States. Despite the identification of many molecular alterations in PDA, this information has not translated into effective therapeutic strategies to date. A recent report in Cancer Cell (Fernandez-Zapico et al., Cancer Cell 2005, 7:39-49) reveals an unexpected role for the hematopoietic-specific RhoGEF VAV1 in pancreatic tumorigenesis, where ectopic expression of VAV1 as a result of promoter demethylation was identified in the majority of established cell lines and PDA tissue samples. Importantly, VAV1 expression was functionally required for optimal proliferation, transformation and survival of pancreatic cancer cell lines. This study provides the first evidence of VAV1 promoter demethylation as an event in cancer progression, suggesting that aberrant signaling pathways driven by VAV1 are potential therapeutic targets in PDA.