Publications

Embracing cancer complexity: Hallmarks of systemic disease

C. Swanton; E. Bernard; C. Abbosh; F. Andre; J. Auwerx et al. 

The last 50 years have witnessed extraordinary developments in understanding mechanisms of carcinogenesis, synthesized as the hallmarks of cancer. Despite this logical framework, our understanding of the molecular basis of systemic manifestations and the underlying causes of cancer-related death remains incomplete. Looking forward, elucidating how tumors interact with distant organs and how multifaceted environmental and physiological parameters impinge on tumors and their hosts will be crucial for advances in preventing and more effectively treating human cancers. In this perspective, we discuss complexities of cancer as a systemic disease, including tumor initiation and promotion, tumor micro- and immune macro-environments, ag ing, metabolism and obesity, cancer cachexia, circadian rhythms, nervous system interactions, tumorrelated thrombosis, and the microbiome. Model systems incorporating human genetic variation will be essential to decipher the mechanistic basis of these phenomena and unravel gene -environment interactions, providing a modern synthesis of molecular oncology that is primed to prevent cancers and improve patient quality of life and cancer outcomes.

Bispecific PD1-IL2v and anti-PD-L1 break tumor immunity resistance by enhancing stem-like tumor- reactive CD8+T cells and reprogramming macrophages

M. Tichet; S. Wullschleger; A. Chryplewicz; N. Fournier; R. Marcone et al. 

Immunotherapies have shown remarkable, albeit tumor-selective, therapeutic benefits in the clinic. Most pa-tients respond transiently at best, highlighting the importance of understanding mechanisms underlying resistance. Herein, we evaluated the effects of the engineered immunocytokine PD1-IL2v in a mouse model of de novo pancreatic neuroendocrine cancer that is resistant to checkpoint and other immunotherapies. PD1-IL2v utilizes anti-PD-1 as a targeting moiety fused to an immuno-stimulatory IL-2 cytokine variant (IL2v) to precisely deliver IL2v to PD-1+ T cells in the tumor microenvironment. PD1-IL2v elicited substantial infiltration by stem-like CD8+ T cells, resulting in tumor regression and enhanced survival in mice. Combining anti-PD-L1 with PD1-IL2v sustained the response phase, improving therapeutic efficacy both by reprogram-ming immunosuppressive tumor-associated macrophages and enhancing T cell receptor (TCR) immune repertoire diversity. These data provide a rationale for clinical trials to evaluate the combination therapy of PD1-IL2v and anti-PD-L1, particularly in immunotherapy-resistant tumors infiltrated with PD-1+ stem-like T cells.

Cancer hallmarks intersect with neuroscience in the tumor microenvironment

D. Hanahan; M. Monje 

The mechanisms underlying the multistep process of tumorigenesis can be distilled into a logical framework involving the acquisition of functional capabilities, the so-called hallmarks of cancer, which are collectively envisaged to be necessary for malignancy. These capabilities, embodied both in transformed cancer cells as well as in the heterotypic accessory cells that together constitute the tumor microenvironment (TME), are conveyed by certain abnormal characteristics of the cancerous phenotype. This perspective discusses the link between the nervous system and the induction of hallmark capabilities, revealing neurons and neuronal projections (axons) as hallmark-inducing constituents of the TME. We also discuss the autocrine and paracrine neuronal regulatory circuits aberrantly activated in cancer cells that may constitute a distinctive “enabling”characteristic contributing to the manifestation of hallmark functions and consequent cancer pathogenesis.

Aberrant hyperexpression of the RNA binding protein FMRP in tumors mediates immune evasion

Q. Zeng; S. Saghafinia; A. A. Chryplewicz; N. Fournier; L. Christe et al. 

Many human cancers manifest the capability to circumvent attack by the adaptive immune system. In this work, we identified a component of immune evasion that involves frequent up-regulation of fragile X mental retardation protein (FMRP) in solid tumors. FMRP represses immune attack, as revealed by cancer cells engineered to lack its expression. FMRP-deficient tumors were infiltrated by activated T cells that impaired tumor growth and enhanced survival in mice. Mechanistically, FMRP’s immunosuppression was multifactorial, involving repression of the chemoattractant C-C motif chemokine ligand 7 (CCL7) concomitant with up-regulation of three immunomodulators-interleukin-33 (IL-33), tumor-secreted protein S (PROS1), and extracellular vesicles. Gene signatures associate FMRP’s cancer network with poor prognosis and response to therapy in cancer patients. Collectively, FMRP is implicated as a regulator that orchestrates a multifaceted barrier to antitumor immune responses.

Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

A. Chryplewicz; J. Scotton; M. Tichet; A. Zomer; K. Shchors et al. 

Glioblastoma (GBM) is poorly responsive to therapy and invariably lethal. One conceivable strategy to circumvent this intractability is to co-target distinctive mechanistic components of the disease, aiming to concomitantly disrupt multiple capabilities required for tumor progression and therapeutic resistance. We assessed this concept by combining vascular endothelial growth factor (VEGF) pathway inhibitors that remodel the tumor vasculature with the tricyclic antidepressant imipramine, which enhances autophagy in GBM cancer cells and unexpectedly reprograms immunosuppressive tumor-associated macrophages via in-hibition of histamine receptor signaling to become immunostimulatory. While neither drug is efficacious as monotherapy, the combination of imipramine with VEGF pathway inhibitors orchestrates the infiltration and activation of CD8 and CD4 T cells, producing significant therapeutic benefit in several GBM mouse models. Inclusion up front of immune-checkpoint blockade with anti-programmed death-ligand 1 (PD-L1) in eventually relapsing tumors markedly extends survival benefit. The results illustrate the potential of mech-anism-guided therapeutic co-targeting of disparate biological vulnerabilities in the tumor microenvironment.

PD-1-cis IL-2R agonism yields better effectors from stem-like CD8(+) T cells

L. C. Deak; V. Nicolini; M. Hashimoto; M. Karagianni; P. C. Schwalie et al. 

Expansion and differentiation of antigen-experienced PD-1(+)TCF-1(+) stem-like CD8(+) T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade(1-4). Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of ‘better effector’ CD8(+) T cells similar to those generated in an acute infection(5). IL-2 binding to the IL-2 receptor alpha-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor beta- and gamma-chain (IL-2R beta gamma)-biased agonists are currently being developed(6-10). Here we show that IL-2R beta gamma-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2R beta gamma on the same cell recovers the ability to differentiate stem-like CD8(+) T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Cancer Cells Retrace a Stepwise Differentiation Program during Malignant Progression

S. Saghafinia; K. Homicsko; A. Di Domenico; S. Wullschleger; A. Perren et al. 

SIGNIFICANCE: Dedifferentiation has long been observed as a histopathologic characteristic of many cancers, albeit inseparable from concurrent increases in cell proliferation. Herein, we demonstrate that dedifferentiation is a mechanistically and temporally separable step in the multistage tumorigenesis of pancreatic islet cells, retracing the developmental lineage of islet beta cells.

Roadmap for the Emerging Field of Cancer Neuroscience

M. Monje; J. C. Borniger; N. J. D’Silva; B. Deneen; P. B. Dirks et al. 

Mounting evidence indicates that the nervous system plays a central role in cancer pathogenesis. In turn, cancers and cancer therapies can alter nervous system form and function. This Commentary seeks to describe the burgeoning field of “cancer neuroscience” and encourage multidisciplinary collaboration for the study of cancer-nervous system interactions.

Myeloid Cells Orchestrate Systemic Immunosuppression, Impairing the Efficacy of Immunotherapy against HPV+ Cancers

G. Galliverti; S. Wullschleger; M. Tichet; D. Murugan; N. Zangger et al. 

Cancers induced by human papillomaviruses (HPV) should be responsive to immunotherapy by virtue of expressing the immunogenic oncoproteins E6/E7. However, advanced forms of cervical cancer, driven by HPV, are poorly responsive to immune response-enhancing treatments involving therapeutic vaccination against these viral neoantigens. Leveraging a transgenic mouse model of HPV-derived cancers, K14HPV16/H2b, we demonstrated that a potent nanoparticle-based E7 vaccine, but not a conventional “liquid” vaccine, induced E7 tumor antigen-specific CD8(+) T cells in cervical tumor-bearing mice. Vaccination alone or in combination with anti-PD-1/anti-CTLA4 did not elicit tumor regression nor increase CD8(+) T cells in the tumor microenvironment (TME), suggesting the presence of immune-suppressive barriers. Patients with cervical cancer have poor dendritic cell functions, have weak cytotoxic lymphocyte responses, and demonstrate an accumulation of myeloid cells in the periphery. Here, we illustrated that myeloid cells in K14HPV16/H2b mice possess potent immunosuppressive activity toward antigen-presenting cells and CD8(+) T cells, dampening antitumor immunity. These immune-inhibitory effects inhibited synergistic effects of combining our oncoprotein vaccine with immune checkpoint-blocking antibodies. Our data highlighted a link between HPV-induced cancers, systemic amplification of myeloid cells, and the detrimental effects of myeloid cells on CD8(+) T-cell activation and recruitment into the TME. These results established immunosuppressive myeloid cells in lymphoid organs as an HPV+ cancer-induced means of circumventing tumor immunity that will require targeted abrogation to enable the induction of efficacious antitumor immune responses.

Synaptic proximity enables NMDAR signalling to promote brain metastasis

Q. Zeng; I. P. Michael; P. Zhang; S. Saghafinia; G. Knott et al. 

Metastasis-the disseminated growth of tumours in distant organs-underlies cancer mortality. Breast-to-brain metastasis (B2BM) is a common and disruptive form of cancer and is prevalent in the aggressive basal-like subtype, but is also found at varying frequencies in all cancer subtypes. Previous studies revealed parameters of breast cancer metastasis to the brain, but its preference for this site remains an enigma. Here we show that B2BM cells co-opt a neuronal signalling pathway that was recently implicated in invasive tumour growth, involving activation by glutamate ligands of N-methyl-D-aspartate receptors (NMDARs), which is key in model systems for metastatic colonization of the brain and is associated with poor prognosis. Whereas NMDAR activation is autocrine in some primary tumour types, human and mouse B2BM cells express receptors but secrete insufficient glutamate to induce signalling, which is instead achieved by the formation of pseudo-tripartite synapses between cancer cells and glutamatergic neurons, presenting a rationale for brain metastasis.

Towards a Cancer Mission in Horizon Europe

A. Berns; U. Ringborg; A. Eggermont; M. Baumann; F. Calvo et al. 

A set of microRNAs coordinately controls tumorigenesis, invasion, and metastasis

I. P. Michael; S. Saghafinia; D. Hanahan 

MicroRNA-mediated gene regulation has been implicated in various diseases, including cancer. This study examined the role of microRNAs (miRNAs) during tumorigenesis and malignant progression of pancreatic neuroendocrine tumors (PanNETs) in a genetically engineered mouse model. Previously, a set of miRNAs was observed to be specifically up-regulated in a highly invasive and metastatic subtype of mouse and human PanNET. Using functional assays, we now implicate different miRNAs in distinct phenotypes: miR-137 stimulates tumor growth and local invasion, whereas the miR-23b cluster enables metastasis. An algorithm, Bio-miRTa, has been developed to facilitate the identification of biologically relevant miRNA target genes and applied to these miRNAs. We show that a top-ranked miR-137 candidate gene, Sorl1, has a tumor suppressor function in primary PanNETs. Among the top targets for the miR-23b cluster, Acvr1c/ALK7 has recently been described to be a metastasis suppressor, and we establish herein that it is down-regulated by the miR-23b cluster, which is crucial for its prometastatic activity. Two other miR-23b targets, Robot and P2ryl, also have demonstrable antimetastatic effects. Finally, we have used the Bio-miRTa algorithm in reverse to identify candidate miRNAs that might regulate activin B, the principal ligand for ALK7, identifying thereby a third family of miRNAs-miRNA-130/301-that is congruently up-regulated concomitant with down-regulation of activin B during tumorigenesis, suggestive of functional involvement in evasion of the proapoptotic barrier. Thus, dynamic up-regulation of miRNAs during multistep tumorigenesis and malignant progression serves to down-regulate distinctive suppressor mechanisms of tumor growth, invasion, and metastasis.

ALK7 Signaling Manifests a Homeostatic Tissue Barrier That Is Abrogated during Tumorigenesis and Metastasis

I. P. Michael; S. Saghafinia; M. Tichet; N. Zangger; I. Marinoni et al. 

Herein, we report that the TGF beta superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and lumina! breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not “authorized” to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand.

Carboplatin/paclitaxel, E7-vaccination and intravaginal CpG as tri-therapy towards efficient regression of genital HPV16 tumors

S. Domingos-Pereira; G. Galliverti; D. Hanahan; D. Nardelli-Haefliger 

High-risk human papillomavirus (HPV) are responsible for genital and oral cancers associated with the expression of the E6/E7 HPV oncogenes. Therapeutic vaccines targeting those oncogenes can only partially control tumor progression, highlighting the necessity to investigate different treatment strategies. Using the genital orthotopic HPV16 TC-1 model, herein we sequentially investigated in progressively more stringent settings the effects of systemic administration of carboplatin/paclitaxel (C + P) chemotherapy combined with HPV16-E7 synthetic long peptide (E7LP) vaccination, followed by intravaginal immunostimulation with the synthetic toll-like-receptor-9 agonist CpG. Our data show that systemic delivery of C+ P prior to E7LP vaccination significantly increased mice survival. This survival benefit was associated with both reduced genital tumor growth at the time of vaccination, and a decreased infiltration of Ly6G myeloid cells and tumor-associated macrophages. Adding intravaginal CpG, which results in increased E7-specific CD8 T cells locally, to E7LP vaccination and the chemotherapy formed a tri-therapy, which significantly increased mice survival as compared to any of the dual treatments. When the tri-therapy was further refined by using a recently optimized nanoparticle-conjugated E7LP vaccine, even larger end-stage genital-TC-1 tumors responded, with 90% of mice showing a survival benefit as compared to 30% of mice with the tri-therapy involving the traditional E7LP ‘liquid’ vaccine. C + P is commonly used to treat cervical cancer patients and its combination with E7/E6 vaccination is currently being tested in a phase I/II trial (NCT02128126). Our data suggests that new vaccine formulations combined with local immunostimulation and standard-of-care chemotherapy have promise to further benefit patients with HPV-associated cancer.

Pan-Cancer Landscape of Aberrant DNA Methylation across Human Tumors

S. Saghafinia; M. Mina; N. Riggi; D. Hanahan; G. Ciriello 

The discovery of cancer-associated alterations has primarily focused on genetic variants. Nonetheless, altered epigenomes contribute to deregulate transcription and promote oncogenic pathways. Here, we designed an algorithmic approach (RESET) to identify aberrant DNA methylation and associated cis-transcriptional changes across >6,000 human tumors. Tumors exhibiting mutations of chromatin remodeling factors and Wnt signaling displayed DNA methylation instability, characterized by numerous hyper- and hypo-methylated loci. Most silenced and enhanced genes coalesced in specific pathways including apoptosis, DNA repair, and cell metabolism. Cancer-germ line antigens (CG) were frequently epigenomically enhanced and their expression correlated with response to anti-PD-1, but not anti-CTLA4, in skin melanoma. Finally, we demonstrated the potential of our approach to explore DNA methylation changes in pediatric tumors, which frequently lack genetic drivers and exhibit epigenomic modifications. Our results provide a pan-cancer map of aberrant DNA methylation to inform functional and therapeutic studies.

Nanoparticle Conjugation of Human Papillomavirus 16 E7-long Peptides Enhances Therapeutic Vaccine Efficacy against Solid Tumors in Mice

G. Galliverti; M. Tichet; S. Domingos-Pereira; S. Hauert; D. Nardelli-Haefliger et al. 

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8(+) T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8(+) T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8(+) T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP- E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a “solid-phase” antigen delivery strategy that is more effective than a conventional free-peptide (“liquid”) vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. (C) 2018 AACR.

GKAP Acts as a Genetic Modulator of NMDAR Signaling to Govern Invasive Tumor Growth

L. Li; Q. Zeng; A. Bhutkar; J. Galvan; E. Karamitopoulou et al. 

A Subset of Cancer-Associated Fibroblasts Determines Therapy Resistance

J. Huelsken; D. Hanahan 

Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma

M. Fankhauser; M. A. S. Broggi; L. Potin; N. Bordry; L. Jeanbart et al. 

In melanoma, vascular endothelial growth factor-C (VEGF-C) expression and consequent lymphangiogenesis correlate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggesting that lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressed this concept in mouse melanoma models with VEGF receptor-3 (VEGFR-3)-blocking antibodies and unexpectedly found that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further found that this effect was mediated by VEGF-C-induced CCL21 and tumor infiltration of naive T cells before immunotherapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, gene expression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrations associated with both T cell activation and expansion after peptide vaccination and clinical response to checkpoint blockade. We propose that VEGF-C potentiates immunotherapy by attracting naive T cells, which are locally activated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarker for immunotherapy response.

The consensus molecular subtypes of colorectal cancer

J. Guinney; R. Dienstmann; X. Wang; A. De Reynies; A. Schlicker et al. 

Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and evident metabolic dysregulation; and CMS4 (mesenchymal, 23%), prominent transforming growth factor-beta activation, stromal invasion and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intratumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC-with clear biological interpretability-and the basis for future clinical stratification and subtype-based targeted interventions.

A Cross-Species Analysis in Pancreatic Neuroendocrine Tumors Reveals Molecular Subtypes with Distinctive Clinical, Metastatic, Developmental, and Metabolic Characteristics

A. Sadanandam; S. Wullschleger; C. A. Lyssiotis; C. Groetzinger; S. Barbi et al. 

Seeking to assess the representative and instructive value of an engineered mouse model of pancreatic neuroendocrine tumors (PanNET) for its cognate human cancer, we profiled and compared mRNA and miRNA transcriptomes of tumors from both. Mouse PanNET tumors could be classified into two distinctive subtypes, well-differentiated islet/insulinoma tumors (IT) and poorly differentiated tumors associated with liver metastases, dubbed metastasis-like primary (MLP). Human PanNETs were independently classified into these same two subtypes, along with a third, specific gene mutation-enriched subtype. The MLP subtypes in human and mouse were similar to liver metastases in terms of miRNA and mRNA transcriptome profiles and signature genes. The human/mouse MLP subtypes also similarly expressed genes known to regulate early pancreas development, whereas the IT subtypes expressed genes characteristic of mature islet cells, suggesting different tumorigenesis pathways. In addition, these subtypes exhibit distinct metabolic profiles marked by differential pyruvate metabolism, substantiating the significance of their separate identities. SIGNIFICANCE: This study involves a comprehensive cross-species integrated analysis of multi-omics profiles and histology to stratify PanNETs into subtypes with distinctive characteristics. We provide support for the RIP1-TAG2 mouse model as representative of its cognate human cancer with prospects to better understand PanNET heterogeneity and consider future applications of personalized cancer therapy. (C) 2015 AACR.

Dual Targeting of the Autophagic Regulatory Circuitry in Gliomas with Repurposed Drugs Elicits Cell-Lethal Autophagy and Therapeutic Benefit

K. Shchors; A. Massaras; D. Hanahan 

The associations of tricyclic antidepressants (TCAs) with reduced incidence of gliomas and elevated autophagy in glioma cells motivated investigation in mouse models of gliomagenesis. First, we established that imipramine, a TCA, increased autophagy and conveyed modest therapeutic benefit in tumor-bearing animals. Then we screened clinically approved agents suggested to affect autophagy for their ability to enhance imipramine-induced autophagy-associated cell death. The anticoagulant ticlopidine, which inhibits the purinergic receptor P2Y(12), potentiated imipramine, elevating cAMP, a modulator of autophagy, reducing cell viability in culture, and increasing survival in glioma-bearing mice. Efficacy of the combination was obviated by knockdown of the autophagic regulatory gene ATG7, implicating cell-lethal autophagy. This seemingly innocuous combination of TCAs and P2Y(12) inhibitors may have applicability for treating glioma.

Deficiency for the Cysteine Protease Cathepsin L Impairs Myc-Induced Tumorigenesis in a Mouse Model of Pancreatic Neuroendocrine Cancer

N. R. Brindle; J. A. Joyce; F. Rostker; E. R. Lawlor; L. Swigart-Brown et al. 

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycER(TAM); Bcl-x(L) model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycER(TAM); Bcl-x(L) tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.

Colorectal cancer classification based on gene expression is not associated with FOLFIRI response Reply

A. Sadanandam; J. Gray; D. Hanahan 

Reconciliation of classification systems defining molecular subtypes of colorectal cancer

A. Sadanandam; X. Wang; F. De Sousa E Melo; J. W. Gray; L. Vermeulen et al. 

Recently we published two independent studies describing novel gene expression-based classifications of colorectal cancer (CRC). Notably, each study stratified CRC into a different number of subtypes: one reported 3 subtypes, whereas the second highlighted 5. Given that each ascribed clinical significance, distinctive biology, and therapeutic prognosis to the different subtypes, we sought to reconcile this apparent incongruity in subtype stratification of CRC, and to interrelate the results. To do so, we each evaluated the other’s data sets and analytical methods and discovered that the subtypes and their classifiers are, in fact, clearly related to each other; indeed, the 5 subtype outcomes can be coalesced into the same three. In addition to presenting this clarification, we briefly discuss how both classification methods can be viewed within the broader literature on CRC subtypes, and potentially applied.

Rethinking the war on cancer

D. Hanahan 

Some 40 years ago a metaphor was posed that cancer was such an insidious adversary that a declaration of war on the disease was justified. Although this statement was a useful inspiration for enlistment of resources, despite extraordinary progress in our understanding of disease pathogenesis, in most cases and for most forms of cancer this war has not been won. A second metaphor was about magic bullets-targeted therapies based on knowledge of mechanisms that were envisaged to strike with devastating consequences for the disease. The reality, however, is that targeted therapies are generally not curative or even enduringly effective, because of the adaptive and evasive resistance strategies developed by cancers under attack. In this Series paper, I suggest that, much like in modern warfare, the war on cancer needs to have a battlespace vision.

A New Twist on Radiation Oncology: Low-Dose Irradiation Elicits Immunostimulatory Macrophages that Unlock Barriers to Tumor Immunotherapy

M. De Palma; G. Coukos; D. Hanahan 

Tumor-infiltrating macrophages typically promote angiogenesis while suppressing antitumoral T cell responses. In this issue of Cancer Cell, Klug and colleagues report that clinically-feasible, low-dose irradiation redirects macrophage differentiation from a tumor-promoting/immunosuppressive state to one that enables cytotoxic T cells to infiltrate tumors and kill cancer cells, rendering immunotherapy successful in mice.

A colorectal cancer classification system that associates cellular phenotype and responses to therapy

A. Sadanandam; C. A. Lyssiotis; K. Homicsko; E. A. Collisson; W. J. Gibb et al. 

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise, individualized treatment strategies are needed. To that end, we analyzed gene expression profiles from 1,290 CRC tumors using consensus-based unsupervised clustering. The resultant clusters were then associated with therapeutic response data to the epidermal growth factor receptor–targeted drug cetuximab in 80 patients. The results of these studies define six clinically relevant CRC subtypes. Each subtype shares similarities to distinct cell types within the normal colon crypt and shows differing degrees of ‘stemness’ and Wnt signaling. Subtype-specific gene signatures are proposed to identify these subtypes. Three subtypes have markedly better disease-free survival (DFS) after surgical resection, suggesting these patients might be spared from the adverse effects of chemotherapy when they have localized disease. One of these three subtypes, identified by filamin A expression, does not respond to cetuximab but may respond to cMET receptor tyrosine kinase inhibitors in the metastatic setting. Two other subtypes, with poor and intermediate DFS, associate with improved response to the chemotherapy regimen FOLFIRI1 in adjuvant or metastatic settings. Development of clinically deployable assays for these subtypes and of subtype-specific therapies may contribute to more effective management of this challenging disease.

Hijacking the Neuronal NMDAR Signaling Circuit to Promote Tumor Growth and Invasion

L. Li; D. Hanahan 

Glutamate and its receptor N-methyl-D-aspartate receptor (NMDAR) have been associated with cancer, although their functions are not fully understood. Herein, we implicate glutamate-driven NMDAR signaling in a mouse model of pancreatic neuroendocrine tumorigenesis (PNET) and in selected human cancers. NMDAR was upregulated at the periphery of PNET tumors, particularly invasive fronts. Moreover, elevated coexpression of NMDAR and glutamate exporters correlated with poor prognosis in cancer patients. Treatment of a tumor-derived cell line with NMDAR antagonists impaired cancer cell proliferation and invasion. Flow conditions mimicking interstitial fluid pressure induced autologous glutamate secretion, activating NMDAR and its downstream MEK-MAPK and CaMK effectors, thereby promoting invasiveness. Congruently, pharmacological inhibition of NMDAR in mice with PNET reduced tumor growth and invasiveness. Therefore, beyond its traditional role in neurons, NMDAR may be activated in human tumors by fluid flow consequent to higher interstitial pressure, inducing an autocrine glutamate signaling circuit with resultant stimulation of malignancy.

Using a preclinical mouse model of high-grade astrocytoma to optimize p53 restoration therapy

K. Shchors; A. I. Persson; F. Rostker; T. Tihan; N. Lyubynska et al. 

Increased invasiveness of MMP-9-deficient tumors in two mouse models of neuroendocrine tumorigenesis

K. Shchors; H. Nozawa; J. Xu; F. Rostker; L. Swigart-Brown et al. 

Despite their apparent success in pre-clinical trials, metalloproteinase (MMP) inhibitors proved to be inefficacious in clinical settings. In an effort to understand the underlying causes of this unanticipated outcome, we modeled the consequences of long-term MMP inhibition by removing one of the major players in tumorigenesis, MMP9, in two complimentary mouse models of pancreatic neuroendocrine carcinogenesis: Myc;BclXl and RIP1-Tag2. By employing gel zymography and a fluoregenic solution assay, we first established that MMP9 is expressed and activated in Myc;BclXl tumors in an interleukin-1β-dependent manner. The genetic deletion of MMP9 in Myc;BclXl mice impairs tumor angiogenesis and growth analogous to its absence in the RIP1-Tag2 model. Notably, tumors that developed in the context of MMP9-deficient backgrounds in both models were markedly more invasive than their typical wild-type counterparts, and expressed elevated levels of pro-invasive cysteine cathepsin B. The increased invasion of MMP9-deficient tumors was associated with a switch in the spectrum of inflammatory cells at the tumor margins, involving homing of previously undetected, cathepsin-B expressing CD11b;Gr1-positive cells to the invasive fronts. Thus, plasticity in the tumor inflammatory compartment is partially responsible for changes in the expression pattern of tumor-associated proteases, and may contribute to the compensatory effects observed on MMP inhibition, hence accounting for the heightened tumor progression described in late stage clinical trials.

Bioavailable copper modulates oxidative phosphorylation and growth of tumors

S. Ishida; P. Andreux; C. Poitry-Yamate; J. Auwerx; D. Hanahan 

Copper is an essential trace element, the imbalances of which are associated with various pathological conditions, including cancer, albeit via largely undefined molecular and cellular mechanisms. Here we provide evidence that levels of bioavailable copper modulate tumor growth. Chronic exposure to elevated levels of copper in drinking water, corresponding to the maximum allowed in public water supplies, stimulated proliferation of cancer cells and de novo pancreatic tumor growth in mice. Conversely, reducing systemic copper levels with a chelating drug, clinically used to treat copper disorders, impaired both. Under such copper limitation, tumors displayed decreased activity of the copper-binding mitochondrial enzyme cytochrome c oxidase and reduced ATP levels, despite enhanced glycolysis, which was not accompanied by increased invasiveness of tumors. The antiproliferative effect of copper chelation was enhanced when combined with inhibitors of glycolysis. Interestingly, larger tumors contained less copper than smaller tumors and exhibited comparatively lower activity of cytochrome c oxidase and increased glucose uptake. These results establish copper as a tumor promoter and reveal that varying levels of copper serves to regulate oxidative phosphorylation in rapidly proliferating cancer cells inside solid tumors. Thus, activation of glycolysis in tumors may in part reflect insufficient copper bioavailability in the tumor microenvironment.

A Biocompatible in Vivo Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice

A. Godinat; H. M. Park; S. C. Miller; K. Cheng; D. Hanahan et al. 

The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin, substrate for firefly luciferase. The success of this “split luciferin” ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a “caged” luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH2-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting in vivo.

Identification and Characterization of Poorly Differentiated Invasive Carcinomas in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis

K. E. Hunter; M. L. Quick; A. Sadanandam; D. Hanahan; J. A. Joyce 

Pancreatic neuroendocrine tumors (PanNETs) are a relatively rare but clinically challenging tumor type. In particular, high grade, poorly-differentiated PanNETs have the worst patient prognosis, and the underlying mechanisms of disease are poorly understood. In this study we have identified and characterized a previously undescribed class of poorly differentiated PanNETs in the RIP1-Tag2 mouse model. We found that while the majority of tumors in the RIP1-Tag2 model are well-differentiated insulinomas, a subset of tumors had lost multiple markers of beta-cell differentiation and were highly invasive, leading us to term them poorly differentiated invasive carcinomas (PDICs). In addition, we found that these tumors exhibited a high mitotic index, resembling poorly differentiated (PD)-PanNETs in human patients. Interestingly, we identified expression of Id1, an inhibitor of DNA binding gene, and a regulator of differentiation, specifically in PDIC tumor cells by histological analysis. The identification of PDICs in this mouse model provides a unique opportunity to study the pathology and molecular characteristics of PD-PanNETs.

Accessories to the Crime: Functions of Cells Recruited to the Tumor Microenvironment

D. Hanahan; L. M. Coussens 

Mutationally corrupted cancer (stem) cells are the driving force of tumor development and progression. Yet, these transformed cells cannot do it alone. Assemblages of ostensibly normal tissue and bone marrow-derived (stromal) cells are recruited to constitute tumorigenic microenvironments. Most of the hallmarks of cancer are enabled and sustained to varying degrees through contributions from repertoires of stromal cell types and distinctive subcell types. Their contributory functions to hallmark capabilities are increasingly well understood, as are the reciprocal communications with neoplastic cancer cells that mediate their recruitment, activation, programming, and persistence. This enhanced understanding presents interesting new targets for anticancer therapy.

Imaging guided trials of the angiogenesis inhibitor sunitinib in mouse models predict efficacy in pancreatic neuroendocrine but not ductal carcinoma

P. Olson; G. C. Chu; S. R. Perry; O. Nolan-Stevaux; D. Hanahan 

Preclinical trials in mice represent a critical step in the evaluation of experimental therapeutics. Genetically engineered mouse models (GEMMs) represent a promising platform for the evaluation of drugs, particularly those targeting the tumor microenvironment. We evaluated sunitinib, an angiogenesis inhibitor that targets VEGF and PDGF receptor signaling, in two GEMMs of pancreatic cancer. Sunitinib did not reduce tumor burden in pancreatic ductal adenocarcinoma (PDAC), whereas tumor burden was reduced in the pancreatic neuroendocrine tumor (PNET) model, the latter results confirming and extending previous studies. To explore the basis for the lack of pathologic response in PDAC, we used noninvasive microbubble contrast-enhanced ultrasound imaging, which revealed that sunitinib reduced blood flow both in PDAC and in PNET, concomitant with a reduction in vessel density; nevertheless, PDAC tumors continued to grow, whereas PNET were growth impaired. These results parallel the response in humans, where sunitinib recently garnered FDA and European approval in PNET, whereas two antiangiogenic drugs failed to demonstrate efficacy in PDAC clinical trials. The demonstration of on-target activity but with discordant benefit in the PDAC and PNET GEMMs illustrates the potential value of linked preclinical and clinical trials.

TRANSLATIONAL MEDICINE Cancer lessons from mice to humans

D. Tuveson; D. Hanahan 

Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy

E. A. Collisson; A. Sadanandam; P. Olson; W. J. Gibb; M. Truitt et al. 

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease. Overall survival is typically 6 months from diagnosis(1). Numerous phase 3 trials of agents effective in other malignancies have failed to benefit unselected PDA populations, although patients do occasionally respond. Studies in other solid tumors have shown that heterogeneity in response is determined, in part, by molecular differences between tumors. Furthermore, treatment outcomes are improved by targeting drugs to tumor subtypes in which they are selectively effective, with breast(2) and lung(3) cancers providing recent examples. Identification of PDA molecular subtypes has been frustrated by a paucity of tumor specimens available for study. We have overcome this problem by combined analysis of transcriptional profiles of primary PDA samples from several studies, along with human and mouse PDA cell lines. We define three PDA subtypes: classical, quasimesenchymal and exocrine-like, and we present evidence for clinical outcome and therapeutic response differences between them. We further define gene signatures for these subtypes that may have utility in stratifying patients for treatment and present preclinical model systems that may be used to identify new subtype specific therapies.

Endogenous Myc maintains the tumor microenvironment

N. M. Sodir; L. B. Swigart; A. N. Karnezis; D. Hanahan; G. I. Evan et al. 

The ubiquitous deregulation of Myc in human cancers makes it an intriguing therapeutic target, a notion supported by recent studies in Ras-driven lung tumors showing that inhibiting endogenous Myc triggers ubiquitous tumor regression. However, neither the therapeutic mechanism nor the applicability of Myc inhibition to other tumor types driven by other oncogenic mechanisms is established. Here, we show that inhibition of endogenous Myc also triggers ubiquitous regression of tumors in a simian virus 40 (SV40)-driven pancreatic islet tumor model. Such regression is presaged by collapse of the tumor microenvironment and involution of tumor vasculature. Hence, in addition to its diverse intracellular roles, endogenous Myc serves an essential and nonredundant role in coupling diverse intracellular oncogenic pathways to the tumor microenvironment, further bolstering its credentials as a pharmacological target.

Brivanib, a Dual FGF/VEGF Inhibitor, Is Active Both First and Second Line against Mouse Pancreatic Neuroendocrine Tumors Developing Adaptive/Evasive Resistance to VEGF Inhibition

E. Allen; I. B. Walters; D. Hanahan 

Purpose: Preclinical trials of a mouse model of pancreatic neuroendocrine tumors (PNET) were conducted to determine whether dual FGF/VEGF pathway inhibition with brivanib can improve first-line efficacy in comparison with VEGF inhibitors lacking fibroblast growth factor (FGF)-inhibitory activity and to characterize second-line brivanib activity before and after the onset of evasive resistance to VEGF-selective therapy.

Pericytes promote endothelial cell survival through induction of autocrine VEGF-A signaling and Bcl-w expression

M. Franco; P. Roswall; E. Cortez; D. Hanahan; K. Pietras 

Endothelial cells (ECs) in blood vessels under formation are stabilized by the recruitment of pericytes, both in normal tissues and during angiogenesis in pathologic situations, including neoplasia. In the tumor vasculature, besides supporting the functionality of blood flow, pericytes protect ECs from antiangiogenic therapies, and have thus been implicated in clinical resistance to vascular targeting drugs. However, the molecular nature of the crosstalk between pericytes and ECs is largely unchartered. Herein, we identified pericyte-induced survival signals in ECs by isolation of vascular fragments derived from tumors that had been genetically or pharmacologically engineered to be either pericyte-rich or pericyte-poor. Pericytes induced the antiapoptotic protein Bcl-w in tumor ECs both in vivo and in vitro, thereby conveying protection from cytotoxic damage. The pericyte-dependent survival signaling in ECs was consequential to enforcement of an autocrine loop involving VEGF-A expression in ECs. Through molecular and functional studies, we delineated a signal transduction pathway in ECs downstream of integrin alpha(v) involving activation of NF-kappa B as the initiating event of the protective crosstalk from pericytes. Our elucidation of pericyte-derived pro-survival signaling in tumor ECs has potentially important implications for clinical development of antiangiogenic drugs, and suggests new therapeutic targets for rational multitargeting of cancer. (Blood. 2011; 118(10): 2906-2917)

Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappa B-Dependent Manner

N. Erez; M. Truitt; P. Olson; D. Hanahan 

Cancer-associated fibroblasts (CAFs) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. We demonstrate that CAFs also mediate tumor-enhancing inflammation. Using a mouse model of squamous skin carcinogenesis, we found a proinflammatory gene signature in CAFs isolated from dysplastic skin. This signature was maintained in CAFs from subsequent skin carcinomas and was evident in mammary and pancreatic tumors in mice and in cognate human cancers. The inflammatory signature was already activated in CAFs isolated from the initial hyperplastic stage in multistep skin tumorigenesis. CAFs from this pathway promoted macrophage recruitment, neovascularization, and tumor growth, activities that are abolished when NF-kappa B signaling was inhibited. Additionally, we show that normal dermal fibroblasts can be “educated” by carcinoma cells to express proinflammatory genes.

Insulin receptor functionally enhances multistage tumor progression and conveys intrinsic resistance to IGF-1R targeted therapy

D. B. Ulanet; D. L. Ludwig; C. R. Kahn; D. Hanahan 

The type 1 insulin-like growth factor receptor (IGF-1R) tyrosine kinase is an important mediator of the protumorigenic effects of IGF-I/II, and inhibitors of IGF-1R signaling are currently being tested in clinical cancer trials aiming to assess the utility of this receptor as a therapeutic target. Despite mounting evidence that the highly homologous insulin receptor (IR) can also convey protumorigenic signals, its direct role in cancer progression has not been genetically defined in vivo, and it remains unclear whether such a role for IR signaling could compromise the efficacy of selective IGF-1R targeting strategies. A transgenic mouse model of pancreatic neuroendocrine carcinogenesis engages the IGF signaling pathway, as revealed by its dependence on IGF-II and by accelerated malignant progression upon IGF-1R overexpression. Surprisingly, preclinical trials with an inhibitory monoclonal antibody to IGF-1R did not significantly impact tumor growth, prompting us to investigate the involvement of IR. The levels of IR were found to be significantly up-regulated during multistep progression from hyperplastic lesions to islet tumors. Its functional involvement was revealed by genetic disruption of the IR gene in the oncogene-expressing pancreatic beta cells, which resulted in reduced tumor burden accompanied by increased apoptosis. Notably, the IR knockout tumors now exhibited sensitivity to anti-IGF1R therapy; similarly, high IR to IGF-1R ratios demonstrably conveyed resistance to IGF-1R inhibition in human breast cancer cells. The results predict that elevated IR signaling before and after treatment will respectively manifest intrinsic and adaptive resistance to anti-IGF-1R therapies.

Survival Benefit With Proapoptotic Molecular and Pathologic Responses From Dual Targeting of Mammalian Target of Rapamycin and Epidermal Growth Factor Receptor in a Preclinical Model of Pancreatic Neuroendocrine Carcinogenesis

C. W. Chiu; H. Nozawa; D. Hanahan 

Purpose Pancreatic neuroendocrine tumors (PNETs), although rare, often metastasize, such that surgery, the only potentially curative therapy, is not possible. There is no effective systemic therapy for patients with advanced PNETs. Therefore, new strategies are needed. Toward that end, we investigated the potential benefit of dual therapeutic targeting of the epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) kinases, using a preclinical mouse model of PNET.

Genetic Deletion of the Desmosomal Component Desmoplakin Promotes Tumor Microinvasion in a Mouse Model of Pancreatic Neuroendocrine Carcinogenesis

M. G. H. Chun; D. Hanahan 

We used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to profile the transcriptome of pancreatic neuroendocrine tumors (PNET) that were either non-invasive or highly invasive, seeking to identify pro- and anti-invasive molecules. Expression of multiple components of desmosomes, structures that help maintain cellular adhesion, was significantly reduced in invasive carcinomas. Genetic deletion of one of these desmosomal components, desmoplakin, resulted in increased local tumor invasion without affecting tumor growth parameters in RT2 PNETs. Expression of cadherin 1, a component of the adherens junction adhesion complex, was maintained in these tumors despite the genetic deletion of desmoplakin. Our results demonstrate that loss of desmoplakin expression and resultant disruption of desmosomal adhesion can promote increased local tumor invasion independent of adherens junction status.

Polymorphic genetic control of tumor invasion in a mouse model of pancreatic neuroendocrine carcinogenesis

M. G. H. Chun; J-H. Mao; C. W. Chiu; A. Balmain; D. Hanahan 

Cancer is a disease subject to both genetic and environmental influences. In this study, we used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to identify a genetic locus that influences tumor progression to an invasive growth state. RT2 mice inbred into the C57BL/6 (B6) background develop both non-invasive pancreatic neuroendocrine tumors (PNET) and invasive carcinomas with varying degrees of aggressiveness. In contrast, RT2 mice inbred into the C3HeB/Fe (C3H) background are comparatively resistant to the development of invasive tumors, as are RT2 C3HB6(F1) hybrid mice. Using linkage analysis, we identified a 13-Mb locus on mouse chromosome 17 with significant linkage to the development of highly invasive PNETs. A gene residing in this locus, the anaplastic lymphoma kinase (Alk), was expressed at significantly lower levels in PNETs from invasion-resistant C3H mice compared with invasion-susceptible B6 mice, and pharmacological inhibition of Alk led to reduced tumor invasiveness in RT2 B6 mice. Collectively, our results demonstrate that tumor invasion is subject to polymorphic genetic control and identify Alk as a genetic modifier of invasive tumor growth.

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

P. Olson; J. Lu; H. Zhang; A. Shai; M. G. Chun et al. 

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.

Increased expression of GAD65 and GABA in pancreatic beta-cells impairs first-phase insulin secretion

Y. Shi; J. Kanaani; V. Menard-Rose; Y. Ma; P. Chang et al. 

The functional role of glutamate decarboxylase (GAD) and its product GABA in pancreatic islets has remained elusive. Mouse β-cells express the larger isoform GAD67, whereas human islets express only the smaller isoform GAD65. We have generated two lines of transgenic mice expressing human GAD65 in pancreatic β-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in β-cells was Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance, whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired glucose tolerance and inhibition of insulin secretion in vivo in response to glucose. In addition, fasting levels of blood glucose are elevated and insulin is decreased in Line 1 homozygotes. Pancreas perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to glucose by affecting a step proximal to or at the KATP +channel.

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase

S. Kash; R. Johnson; L. Tecott; J. Noebels; R. Mayfield et al. 

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67−/− animals, which are born with developmental abnormalities and die shortly after birth, GAD65−/− mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5′ phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65−/− mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65−/− mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65−/− mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65−/− animals as a model of epilepsy involving GABA-ergic pathways.

Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction

S-M. Kang; D. B. Schneider; Z. Lin; D. Hanahan; D. A. Dichek et al. 

Fas ligand is believed to mediate immune privilege in a variety of tissues, including the eye, testis, and a subset of tumors. We tested whether expression of Fas ligand on pancreatic islets either following adenovirai or germline gene transfer could confer immune privilege after transplantation. Islets were infected with an adenovirai vector containing the murine Fas ligand cDNA (AdFasL), and were transplanted into allogenic diabetic hosts. Paradoxically, AdFasL-infected islets underwent accelerated neutrophilic rejection. The rejection was T cell and B cell independent and required Fas protein expression by host cells, but not on islets. Similarly, transgenic mice expressing Fas ligand in pancreatic beta cells developed massive neutrophilic infiltrates and diabetes at a young age. Thus, Fas ligand expression on pancreatic islets results in neutrophilic infiltration and islet destruction. These results have important implications for the development of Fas ligand-based immunotherapies.

Co-targeting autophagy, macrophages and vasculature in glioma tumors triggers tumor immunity.

A. Chryplewicz; J. Scotton; M. Tichet; D. Hanahan 

Reprogramming immunosuppressive tumor-associated macrophages potentiates standard-of-care therapy in melanoma

M. Tichet; A. Chryplewicz; D. Hanahan 

Co-targeting distinct hallmark capabilities for therapeutic benefit in pre-clinical GBM models

A. Chryplewicz; D. Hanahan 

The immunocytokine PD1-IL2v overcomes immune checkpoint resistance, and combination with an anti-PD-L1 antibody further enhances its anti-tumor activity.

S. Wullschleger; M. Tichet; L. Codarri-Deak; P. Umana; C. Klein et al. 

A personalised approach for anti-GITR-based immunotherapy in preclinical models of pancreatic ductal adenocarcinoma

K. Desai; C. Ragulan; P. V. Lawrence; S. Wullschleger; M. L. Tichet et al. 

Unsorted single-cell RNA sequencing profiles of metastatic melanoma patients reveal the heterogeneity of melanoma-associated fibroblasts

K. Homicsko; D. Barras; V. A. Lopez; B. G. Moura; G. Berthod et al. 

Proton pump inhibitors negatively impact survival of PD-1 inhibitor based therapies in metastatic melanoma patients

K. Homicsko; G. Richtig; F. Tuchmann; Z. Tsourti; D. Hanahan et al. 

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

A. J. O’Donoghue; S. L. Ivry; C. Chaudhury; D. R. Hostetter; D. Hanahan et al. 

The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only -procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.

Hallmarks of cancer: applications to cancer medicine?

D. Hanahan 

Time-resolved imaging system for fluorescence-guided surgery with lifetime imaging capability

F. Powolny; K. Homicsko; R. Sinisi; C. Bruschini; E. Grigoriev et al. 

We present a single-photon camera for fluorescence imaging, with a time resolution better than 100ps, capable of providing both intensity and lifetime images. the camera was fabricated in standard CMOS technology. With this FluoCam we show the possibility to study sub-nanosecond fluorescence mechanisms. The FluoCam was used to characterize a near-infrared probe, indocyanine green, conjugated with multimeric cyclic pentapeptide ( cRGD). The fluorescent probe-conjugated was used to target and mark tumors with better specificity, in particular aiming at targeting the integrins alpha(v)beta(3) and alpha(v)beta(5). As a first step towards clinical studies, preliminary results obtained in-vivo are presented. The first envisioned clinical application would be image-guided surgical oncology to help the surgeon to remove tumor tissue by a better discrimination from normal tissues and also to improve the detection of metastatic lymph nodes. A further application could be the in-vivo determination of the alpha(v)beta(3) and alpha(v)beta(5) targets to select patients for therapy with RGD-chemotherapy conjugates.

Copper chelation therapy suppresses tumor growth by inhibiting mitochondrial ATP production in tumors.

S. Ishida; P. Andreux; J. Auwerx; D. Hanahan 

Hallmarks Of Cancer: A 2012 Perspective

D. Hanahan 

Milestones in tumor vascularization and its therapeutic targeting

M. De Palma; D. Hanahan 

Research into the mechanisms and manifestations of solid tumor vascularization was launched more than 50 years ago with the proposition and experimental demonstrations that angiogenesis is instrumental for tumor growth and was, therefore, a promising therapeutic target. The biological knowledge and therapeutic insights forthcoming have been remarkable, punctuated by new concepts, many of which were not foreseen in the early decades. This article presents a perspective on tumor vascularization and its therapeutic targeting but does not portray a historical timeline. Rather, we highlight eight conceptual milestones, integrating initial discoveries and recent progress and posing open questions for the future.|De Palma and Hanahan outline advances in understanding tumor angiogenesis and discuss the therapeutic opportunities of targeting tumor vascularization.

Cancer Evolution: A Multifaceted Affair

G. Ciriello; L. Magnani; S. J. Aitken; L. Akkari; S. Behjati et al. 

Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery.Significance: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.

Hallmarks of Cancer: New Dimensions

D. Hanahan 

The hallmarks of cancer conceptualization is a heuristic tool for distilling the vast complexity of cancer phenotypes and genotypes into a provisional set of underl y-ing principles. As knowledge of cancer mechanisms has progressed, other facets of the disease have emerged as potential refi nements. Herein, the prospect is raised that phenotypic plasticity and dis-rupted differentiation is a discrete hallmark capability, and that nonmutational epigenetic reprogram-ming and polymorphic microbiomes both constitute distinctive enabling characteristics that facilitate the acquisition of hallmark capabilities. Additionally, senescent cells, of varying origins, may be added to the roster of functionally important cell types in the tumor microenvironment. Significance: Cancer is daunting in the breadth and scope of its diversity, spanning genetics, cell and tissue biology, pathology, and response to therapy. Ever more powerful experimental and computa-tional tools and technologies are providing an avalanche of “big data” about the myriad manifestations of the diseases that cancer encompasses. The integrative concept embodied in the hallmarks of cancer is helping to distill this complexity into an increasingly logical science, and the provisional new dimen-sions presented in this perspective may add value to that endeavor, to more fully understand mecha-nisms of cancer development and malignant progression, and apply that knowledge to cancer medicine.

The biology of personalized cancer medicine: facing individual complexities underlying hallmark capabilities

M. De Palma; D. Hanahan 

It is a time of great promise and expectation for the applications of knowledge about mechanisms of cancer toward more effective and enduring therapies for human disease. Conceptualizations such as the hallmarks of cancer are providing an organizing principle with which to distill and rationalize the abject complexities of cancer phenotypes and genotypes across the spectrum of the human disease. A countervailing reality, however, involves the variable and often transitory responses to most mechanism-based targeted therapies, returning full circle to the complexity, arguing that the unique biology and genetics of a patient’s tumor will in the future necessarily need to be incorporated into the decisions about optimal treatment strategies, the frontier of personalized cancer medicine. This perspective highlights considerations, metrics, and methods that may prove instrumental in charting the landscape of evaluating individual tumors so to better inform diagnosis, prognosis, and therapy. Integral to the consideration is remarkable heterogeneity and variability, evidently embedded in cancer cells, but likely also in the cell types composing the supportive and interactive stroma of the tumor microenvironment (e.g., leukocytes and fibroblasts), whose diversity in form, regulation, function, and abundance may prove to rival that of the cancer cells themselves. By comprehensively interrogating both parenchyma and stroma of patients’ cancers with a suite of parametric tools, the promise of mechanism-based therapy may truly be realized.

Hallmarks of Cancer: The Next Generation

D. Hanahan; R. A. Weinberg 

The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two emerging hallmarks of potential generality to this list-reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the “tumor microenvironment.” Recognition of the widespread applicability of these concepts will increasingly affect the development of new means to treat human cancer.

Roles of Epigenetic Dysregulation in Cancer Progression and Metastasis

Combinatorial targeting of angiogenesis with inducers of autophagy in the treatment of glioblastoma multiforme

NMDAR signaling in cancer

A pan-cancer classification based on fmrp pathway activity that informs differential prognosis and therapeutic responses

S. Saqafi; D. Hanahan 

The present invention relates to methods and compositions which provide a companion diagnostic for cancer therapy. A method for identifying and stratifying a patient or group of patients with cancer as (i) being high or low for FMRP activity, (ii) having a high or low risk prognosis, and/or (iii) being a responder or non-responder to cancer therapy, and/or (iv) having high or low immune cell infiltrated tumor.

FMRP and cancer treatment

Q. Zeng; D. Hanahan 

The present invention provides compositions and methods for down-modulating the expression and/or the immuno-suppressive activity of i) the FMRP protein, ii) an mRNA encoding the FMRP protein, and/or iii) the FMR1 gene for the treatment and/or prevention of primary cancer and/or cancer metastasis in a subject in need thereof.

Method for determining cancer invasiveness and patient prognosis

D. Hanahan; L. Li; A. Bhutkav 

A method for determining the prognosis for survival of a patient having cancer is disclosed, said cancer being altered (i.e. highly activated or alternatively inactive/inhibited) in the NMDAR signalling pathway, said method comprising determining the expression levels of a plurality of gene products in a sample from a cancer patient relative to a control, wherein the differential expression of said plurality of gene products relative to a control is indicative of cancer aggressiveness and of the patient’s prognosis. A method for improving the prognosis for a cancer patient being diagnosed to have a poor prognosis, as well as pharmaceutical compositions for that purpose, are also disclosed, said method comprising modulating the level or the activity of the identified gene products and/or a member of the NMDA signalling pathways and/or a DLGAP family gene products and/or a HSF1 gene product and/or a FMR1 gene product.

Colorectal cancer classification with differential prognosis and personalized therapeutic responses

A. Sadanandam; C. Lyssiotis; D. Hanahan; J. Gray 

The present invention relates to gene sets, the expression levels of which are useful for classifying colorectal tumors and predicting disease-free prognosis and response of patients to specific therapies that are either novel or currently available in the clinics for colorectal cancer patients.

Regulatory role of miRNAs in metastasis of pancreatic neuroendocrine tumors

M. S. Saghafinia