Dmitriy Kedrin

High-fat diet enhances stemness and tumorigenicity of intestinal progenitors

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we show that high-fat diet (HFD)-induced obesity augments the numbers and function of Lgr5+ intestinal stem cells of the mammalian intestine. Mechanistically, a HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-δ) signature in intestinal stem cells and progenitor cells (non-intestinal stem cells), and pharmacological activation of PPAR-δ recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-δ-dependent manner. Notably, HFD- and agonist-activated PPAR-δ signalling endow organoid-initiating capacity to progenitors, and enforced PPAR-δ signalling permits these progenitors to form in vivo tumours after loss of the tumour suppressor Apc. These findings highlight how diet-modulated PPAR-δ activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumours.

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.

Anti-VEGF therapy induces ECM remodeling and mechanical barriers to therapy in colorectal cancer liver metastases

Anti-VEGF therapy enhances mechanical barriers to therapy in colorectal cancer liver metastases by increasing hyaluronan deposition. Stiff resistance to cancer therapy Antiangiogenic therapy with drugs that block vascular endothelial growth factor (VEGF) signaling to inhibit formation of new blood vessels in tumors is commonly used in colorectal cancer. Unfortunately, the effects of this therapy usually do not last for long, and a study by Rahbari et al. shows why this might be the case. The authors found that VEGF inhibition increased the stiffness of colorectal cancer liver metastases, making them more difficult to treat with chemotherapy. In a mouse model, the researchers were able to overcome this difficulty by using an enzyme to degrade a component of the extracellular matrix in liver metastases, suggesting that the matrix may be a target for future cancer therapies. The survival benefit of anti–vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy.

Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice

An alternate route for metastatic cells Metastatic tumor cells are thought to reach distant organs by traveling through the blood circulation or the lymphatic system. Two studies of mouse models now suggest a hybrid route for tumor cell dissemination. Pereira et al. and Brown et al. used distinct methodologies to monitor the fate of tumor cells in lymph nodes. They found that tumor cells could invade local blood vessels within a node, exit the node by entering the blood circulation, then go on to colonize the lung. Whether this dissemination route occurs in cancer patients is unknown; the answer could potentially change the way that affected lymph nodes are treated in cancer. Science, this issue p. 1403, p. 1408 In mice, tumor cells can metastasize to distant organs by entering blood vessels within the local lymph node. Lymph node metastases in cancer patients are associated with tumor aggressiveness, poorer prognoses, and the recommendation for systemic therapy. Whether cancer cells in lymph nodes can seed distant metastases has been a subject of considerable debate. We studied mice implanted with cancer cells (mammary carcinoma, squamous cell carcinoma, or melanoma) expressing the photoconvertible protein Dendra2. This technology allowed us to selectively photoconvert metastatic cells in the lymph node and trace their fate. We found that a fraction of these cells invaded lymph node blood vessels, entered the blood circulation, and colonized the lung. Thus, in mouse models, lymph node metastases can be a source of cancer cells for distant metastases. Whether this mode of dissemination occurs in cancer patients remains to be determined.

Genetics of the Serrated Pathway to Colorectal Cancer

Accounting for ~15% of all colorectal cancers (CRCs), the serrated pathway represents an alternate mechanism of colorectal carcinogenesis that yields microsatellite stable (MSS) tumors and the overwhelming fraction of “sporadic” microsatellite instability high (MSI-H) tumors. Moreover, the MSI-H tumors derived from the serrated pathway are more common of the two, and frequently display excessive CpG island promoter hypermethylation (CIMP-high; Table 1). This promoter hypermethylation results in epigenetic silencing of a large number of tumor-suppressor genes, including MLH1, which causes the associated MSI-H phenotype. Somatic mutations typically include activating mutations in BRAF (V600E), and less commonly KRAS or aberrant EGFR activation, which occur during the early stages of serrated polyp development. In contrast to tubular adenomas, bilallelic inactivation of APC is not an initiating event in this pathway (Figure 1). Despite this molecular understanding and the development of novel drugs to target them, additional treatments are still needed given the inferior outcomes observed in BRAF-mutated colon cancers within the context of their microsatellite status.1 Figure 1 Serrated Pathway to Colorectal Cancer. Schematic comparing the mutational changes involved from normal mucosa to colorectal cancer. The top half represents the conventional pathway to colorectal cancer, with the most frequent mutational events described. ... Table 1 Important acronyms and definitions Discovery of high-risk genetic variants for this pathway represents a promising strategy to identify additional therapeutic targets for this subset of colorectal cancers. Epidemiologic data from families of serrated polyposis patients strongly suggest a heritable predisposition exists toward serrated colorectal carcinogenesis. First-degree relatives of serrated polyposis patients are at significantly higher elevated risk of developing serrated polyps themselves. In addition, elevated pleiotropic cancer risks are present in these families. An Australian cohort demonstrated that relatives are at an increased risk of pancreatic cancer.2 First-degree relatives of those with MSI-H CRC who do not have Lynch syndrome or serrated polyposis are at increased risk (standardized incidence ratios) of developing stomach, ovarian, and liver cancers.3 Through exome sequencing of individuals who develop multiple sessile serrated polyps and/or serrated polyposis, we recently identified novel high-risk variants for the serrated pathway.4 We demonstrated that such individuals are approximately fourfold enriched for rare, germline loss-of-function (LoF) mutations (defined as nonsense and splice-site mutations) in genes responsible for oncogene-induced senescence (OIS) mechanisms. OIS is a tumor-suppressive mechanism that is activated by the replicative and metabolic stress caused by oncogenic transformation. This hypothesis was based on the observations from genetic mouse models of serrated neoplasia in which the BRAF V600E mutation or activating KRAS mutation was alone sufficient to induce serrated neoplasia in the long-term; however, in the short-term, OIS barriers prevented rapid tumorigenesis.5, 6, 7 Concurrent inactivation of these OIS mechanisms with activating BRAF/KRAS mutations greatly expedited serrated neoplasia formation. In humans, activation of these critical OIS pathways (ATM–ATR DNA damage pathway and p16-RB pathway) has been previously demonstrated to be relevant in colonic precursor lesions in addition to lesions in other tissue types. Several of the OIS genes identified in serrated polyposis patients in this study (ATM, RBL1, and XAF1) have been previously implicated in human or animal studies of colorectal carcinogenesis. We next demonstrated that the remaining patients with serrated polyposis (who do not have an obvious loss-of-function mutation) actually harbor deleterious variants in genes previously unassociated, but critical to these OIS mechanisms. Cross-referencing all rare LoF mutations found in patients with an orthogonal database (not dependent on senescence characteristics) of all genes implicated in cancer by genome-wide association studies, we discovered two unrelated individuals with identical nonsense mutations in RNF43, a gene frequently mutated in mucinous neoplasms of the pancreas and stomach that encodes for a negative regulator of Wnt signaling through Wnt receptor endocytosis. This enrichment in cases of serrated polyposis was significant over controls with sizeable effect sizes (odds ratio 460, P=6.8 × 10−5). Analysis of publicly available microarrays of sporadic serrated polyps and tubular adenomas, we found RNF43 to be significantly downregulated in the serrated pathway. Through functional experiments in pancreatic duct cells harboring the KRAS G12D oncogene, we demonstrated that silencing of RNF43 impaired ATR–ATM DNA damage signaling in response to UV radiation, as evident by impaired phosphorylation of Chk1 and p53. Subsequently, another group has further generalized the importance of RNF43 to the development of sporadic MSI-H colorectal cancers.8 Performing whole-exome sequencing on 185 formalin-fixed, paraffin-embedded colon cancers from two Harvard cohorts, Giannakis et al. discovered deleterious somatic mutations in RNF43 to be present in 18.9% of these cancers, in addition to being frequently mutated in endometrial cancers. Interestingly, 50% of the deleterious mutations discovered were frameshift mutations occurring at microsatellite loci within the gene. Validation of a small subset of tumors by next-generation sequencing or Sanger sequencing demonstrated an overall accuracy of 97% for the RNF43 mutation calls made by software. To replicate these results, the authors reanalyzed 222 colorectal cancer cases from The Cancer Genome Atlas.9 RNF43 mutations were present reliably in 17.6% of cases, and 49 cases were from the initial publication. The discrepancy between these results and previously published analyses of the TCGA data set may be attributable to newer algorithms in the detection of significant insertion and deletion events, which continue to be an ongoing challenge in their accuracy compared with single-nucleotide polymorphisms. Under prior methodologies, many true mutations at microsatellite sites were falsely discarded as errors due to their resemblance to sequencing artifacts caused by polymerase slippage during the exome enrichment step. Consistent with the importance of this RNF43 in the serrated pathway, these mutations were particularly enriched in those tumors with MSI-H status, occurring in ~80% of this subset of colorectal cancers (P<2.2 × 10−16, Fishers exact test). Complementary to our initial microarray comparisons of sporadic polyps from the serrated and conventional pathway of colorectal carcinogenesis, somatic RNF43 mutations appeared mutually exclusive with deleterious APC mutations. The results from these studies have clinical consequences for epidemiology, genetic testing, and treatment strategies. First, these mutations provide firm genetic support for the multiple cancer risks observed in families and first-degree relatives of those afflicted with serrated polyposis. Many of the genes found in the primary mechanisms of OIS contain tumor-suppressor genes with already established pleiotropic effects. The discoveries of germline and somatic RNF43 mutations in serrated lesions provide additional genetic evidence of such pleiotropy as the gene is found frequently mutated in gastric, pancreatic, ovarian, and endometrial cancers. The germline mechanisms disrupted in serrated polyposis patients and the somatic mutations found in sporadic MSI-H tumors should promote clinical trials of newly developed chemotherapeutics for sporadic serrated colorectal tumors. Clinical trials of poly(ADP-ribose) polymerase inhibitors have demonstrated promise in individuals with germline mutations in upstream DNA damage repair pathways, notably those with BRCA1 or BRCA2 mutations, for individuals with breast or ovarian cancers.10, 11 Such agents push tumor cells with deficiencies in DNA repair pathways into mitotic catastrophe with the accumulation of double-stranded DNA breaks. The presence of germline and somatic RNF43 mutations also confers the possibility of using an additional class of therapeutic agents. Recently, porcupine inhibitors, a new class of drugs that impair Wnt secretion, have demonstrated efficacy in slowing the tumor growth of pancreatic cancer cell lines that harbor deleterious somatic RNF43 mutations.12 Despite the initial focus upon epigenetics due to the observed CIMP-high phenotype, the genetics of serrated neoplasia have a critical role in determining disease risk and therapeutic strategies. Additional experiments with larger cohorts of serrated polyposis will likely reveal additional high-risk genes given the broad genetic heterogeneity evident, leading to discovery of additional novel targets. In contrast to other tumor types with hypermethylation, the search for the inciting somatic mechanisms that trigger the widespread epigenetic changes observed in these cancers remains uncertain. These efforts over the upcoming years have the potential to further change preventative and therapeutic approaches toward colon cancer.

Bariatric Surgery Prior to Index Screening Colonoscopy Is Associated With a Decreased Rate of Colorectal Adenomas in Obese Individuals

OBJECTIVES: Obesity is an important risk factor for the development of colorectal cancer (CRC). Although the impact of bariatric surgery on CRC is conflicting, its impact on precursor lesions is unknown. The aim of this study was to determine whether bariatric surgery before index screening colonoscopy is associated with decreased development of colorectal adenomas. METHODS: We performed a retrospective cohort study of bariatric surgery patients who had undergone index, screening colonoscopy at an academic center from 2001 to 2014. Patients who had bariatric surgery at least 1 year before index colonoscopy were compared with those who had surgery after colonoscopy, using multivariable logistic regression to control for presurgical body mass index, sex, gender, race, type of surgery, aspirin use, metformin use, smoking, and age at colonoscopy. RESULTS: One hundred and twenty‐five obese individuals who had bariatric surgery before colonoscopy were compared with 223 individuals who had colonoscopy after surgery. Adenomatous polyps were found in 16.8% of individuals who had surgery first vs. 35.5% who had colonoscopy before bariatric surgery (unadjusted odds ratio (OR) 0.37, 95% confidence interval (CI): 0.21–0.64, P=0.0003). After multivariable adjustment, bariatric surgery before index screening colonoscopy was associated with a decreased risk of adenomas at index colonoscopy (adjusted OR 0.37, 95% CI: 0.19–0.69, P=0.002). CONCLUSIONS: Bariatric surgery is associated with a decreased risk of colorectal adenomas in obese individuals without a family history of CRC.

Author Correction: High-fat diet enhances stemness and tumorigenicity of intestinal progenitors

In Fig. 4e of this Article, the labels for ‘Control’ and ‘HFD’ were reversed (‘Control’ should have been labelled blue rather than purple, and ‘HFD’ should have been labelled purple rather than blue). Similarly, in Fig. 4f of this Article, the labels for ‘V’ and ‘GW’ were reversed (‘V’ should have been labelled blue rather than purple, and ‘GW’ should have been labelled purple instead of blue). The original figure has been corrected online.

Corrigendum: In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis

This corrects the article DOI: 10.1038/nbt.3836

Abstract PR06: VEGF-targeted therapy induces extracellular matrix remodeling and increases mechanical barriers to therapy in colorectal cancer liver metastases

Introduction: The anti-VEGF antibody bevacizumab in combination with chemotherapy is a standard treatment for metastatic colorectal cancer (mCRC) today, based on an overall survival improvement. However, this survival benefit is modest and mCRC ultimately progresses. The underlying mechanism of resistance to anti-angiogenic therapy remains unclear. Recent preclinical studies have shown that anti-angiogenic therapy increases collagen expression in tumors as a consequence of hypoxia. The extracellular matrix (ECM) play an important role in solid stress-induced blood vessel collapse as they transmit the mechanical stress created by proliferating cells within the confined space of a tumor. In this study, we investigated the effects of anti-angiogenic therapy on extracellular matrix expression, both collagenous and non-collagenous and perfusion as a novel mechanism of acquired resistance to anti-angiogenic therapy in liver metastases from CRC. Experimental Design: We used C57BL/6 and BALB/c, as well as CCR2-/- and ATR1-/- mice for this study. To generate liver metastasis, we exteriorized and transected spleen, inject syngenic CRC SL4 or CT26 cells (0.1 million cells) in one hemispleen and then, removed it. The other hemispleen remained intact. We monitored tumor burden by measuring blood Gaussia luciferase (Gluc) activity from Gluc transduced tumors and/or by high-frequency ultrasound imaging. Treatments include anti-VEGF monoclonal antibody B20.4-1.1 (5 mg/kg or 1 mg/kg i.p. 2x/week, Genentech), an anti-Ly6G antibody (5 mg/kg i.p. every 2days), pegylated hyaluronidase (PEGHAse, 4.5 mg/kg i.v. 2x/week 24 hour prior to administration of chemotherapy), 5-Fluorouracil (50 mg/kg i.v. 2x/week). We determined ECM–hyaluronic acid (HA), sulfated glycosaminoglycan (sGAG), and collagen contents—by ELISA, the Blyscan Proteoglycan and Glycosaminoglycan assay, and hydroxyproline assay, respectively. We further determined ECM, activated fibroblasts, vasculature, perfusion (Hechst33342), and hypoxia (pimonidazole) by immunohistochemistry, and immune cell profile using flow cytometry. We then, determined stiffness (Young9s modulus) of tumors by unconfined compression tests (Cancer Res 60: 2497-503, 2000) and solid stress using a recently established method (PNAS 109: 15101-8, 2012). We obtained patient samples from the Heidelberg University Hospital and analyzed surgical specimens of patients who underwent liver resection for colorectal liver metastases without preoperative chemotherapy, after preoperative chemotherapy without bevacizumab or after preoperative chemotherapy with bevacizumab. Results: Here, we show that anti-VEGF therapy induces remodeling of the extracellular matrix with subsequent alteration of physical properties of liver metastases. We find that preoperative treatment with bevacizumab in patients with colorectal liver metastases resulted in a significant increase in HA deposition within the tumor. Moreover, in two syngeneic mouse models of mCRC, we show that anti-VEGF therapy markedly increased expression of HA and sGAG, without significant changes in collagen deposition. The density of these matrix components correlates with tumor stiffness; the latter was also significantly increased after anti-VEGF therapy. In time-course and immunofluorescence studies, treatment-induced hypoxia appeared as the driving force for enhanced extracellular matrix expression. Enzymatic depletion of HA partially reversed compromised perfusion in liver metastases after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and systemic chemotherapy. Conclusion: These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy. This abstract is also being presented as Poster B27. Citation Format: Nuh N. Rahbari, Dmitriy Kedrin, Joao Incio, Hao Liu, William T. Ho, Hadi T. Nia, Christina M. Edrich, Keehoon Jung, Julien Daubriac, Ivy Chen, Takahiro Heishi, John Martin, Yuhui Huang, Nir Maimon, Christoph Reissfelder, Juergen Weitz, Yves Boucher, Jeffrey W. Clark, Alan J. Grodzinsky, Dan G. Duda, Rakesh K. Jain, Dai Fukumura. VEGF-targeted therapy induces extracellular matrix remodeling and increases mechanical barriers to therapy in colorectal cancer liver metastases. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR06.

Abstract P3-01-22: Sentinel lymph node metastases in breast cancer: A contributor to distant metastases?

Breast cancer metastasis remains a major cause of mortality in patients. Although significant progress has been made in understanding the mechanisms of this complex process, the findings have yet to be translated into improved survival rates in patients with metastatic disease. The presence of lymph node metastasis in most breast cancer patients is associated with tumor aggressiveness, poorer prognosis and often results in the need for systemic therapy. However, whether tumor cells in the lymph node exit and contribute to distant metastases remains controversial. To track the fate of tumor cells that metastasized to the lymph node, we engineered breast tumor cell lines to express Dendra2, a photo-convertible protein that fluoresces green in the native state and on light activation converts to red fluorescence. Using a novel chronic lymph node window that allows time-lapse imaging over a period of 10 days, we were able to track the movement of cancer cells in the dynamic microenvironment using high- resolution multi-photon microscopy. Our studies show that tumor cells entering the lymph node through the afferent lymphatic vessel proliferate in the sub-capsular sinus and later begin to invade the lymph node parenchyma. Importantly, by photo-conversion of tumor cells in the lymph node, we are able to track the tumor cells that escaped the lymph node and entered the blood circulation. The circulating tumor cells that transited through the lymph node were viable and proliferated in vitro. Our data indicate that metastatic breast cancer cells can exit the node and potentially colonize distant organs. Citation Format: Pereira ER, Kedrin D, Jones D, Beech E, Taghian A, Padera TP. Sentinel lymph node metastases in breast cancer: A contributor to distant metastases?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-01-22.