Characterizing Inflammatory Links in Liver Cancer

The past few years yielded an explosion of exciting clinical trials showing the remarkable benefit of immune treatments in cancer patients. The link between inflammation and cancer is now established, yet the underlying molecular mechanisms are unresolved. As tumors progress, they modulate the inflammatory cells towards a protumorigenic immunosuppressive phenotype. We have shown that the inflammatory cells reciprocate by sculpting the parenchymal epithelial cells. We are studying these reciprocal interactions and hypothesize that they lie at the heart of the link between inflammation and cancer.

Liver cancer is the third most common cause of cancer-related death worldwide, and its dramatic three decades-long rise makes liver cancer the fastest-growing cause of deaths from cancer in the United States. Liver cancer is a prototype of inflammation induced cancer. We employ several strategies to analyze the changes that occur in inflammatory cells before and after liver tumor emergence, based on our preliminary findings showing that changes in inflammatory cells precede tumorigenesis. We are comprehensively mapping the changing inflammatory microenvironment in mouse models of inflammation induced Hepatocellular carcinoma (HCC) – the most common form of primary liver cancer. Using genetic manipulation strategies, coupled with cell isolation techniques we are delineating the molecular cues that mediate these changes and are analyzing the functional role of key mediators of these processes in the malignant process.

We have recently characterized a new form of inflammation which is characterized by the presence of focal collections of immune cells (also known as ectopic lymphoid like structures). Surprisingly, this form of inflammation promotes cancer growth by hijacking molecules which are secreted by lymphocytes, which are usually associated with anti-tumor responses. We are now testing how the new and exciting immune check point drugs interact with these foci. This could impact immunotherapy of cancer, wherein lymphocytes are usually considered positive mediators of anti-cancer responses.

Lab Alumni

Dr. Udi Udi Gluschnaider, PhD

Dr. Shlomi Finkin, PhD

Dr. Simona Hefetz-Sela, PhD

Dr. David Knigin, MD-PhD

Dr. Ruth Peretz, MD-PhD

Dr. Rinnat Porat, PhD

Dr. Efi Weitman, DMD-PhD

Funding

Israel Science foundation (ISF) Center of Excellence

Adelson Medical Research Foundation (AMRF)

German Cancer Research Center (DKFZ) – Ministry of Science, Technology & Space (MOST) Program in Cancer Research

Immune-dependent liver micro niches foster tumor progenitors before they acquire self-sufficiency

Whereas the innate immune system often promotes carcinogenesis, adaptive immunity is known to have a tumor protective role. On the contrary, using a unique mouse model of HCC, we discovered a novel mechanism through which the adaptive immune system can critically support tumorigenesis. We generated genetically modified mice which express a constitutively active form of IKKβ, the upstream kinase of the NF-kB signaling pathway, specifically in hepatocytes (IKKβ(EE)^Hep mice). The IKKβ(EE)^Hep mouse model of HCC displays abundant hepatic ectopic lymphoid-like structures (ELSs) that are similar to their human equivalents in both cellular and cytokine composition. Inflammation usually entails a diffuse influx of immune cells, scattered throughout the inflamed tissue. However, infiltrating leukocytes often form ectopic lymphoid aggregates or even more complex structures that histologically resemble lymphoid organs. These structures direct various B and T cell responses and are referred to as ELSs. The mechanisms governing ELSs neogenesis, as their functional state in different pathologies, remain poorly defined. ELSs often develop at sites of chronic inflammation where they influence the course of many disease types, including distinct autoimmune, cardiovascular, metabolic and neurodegenerative diseases. Clinically, the presence of ELSs within inflamed tissues has been linked to both protective and deleterious outcomes in patients. In cancer the presence of tumor-associated ELSs usually correlates with a better prognosis and they are thought to coordinate endogenous antitumor immune responses (e.g. in melanoma, colorectal and breast cancer). Yet, in HCC we recently revealed a surprising protumorigenic role for ELSs, and found that they constitute immunopathological micro niches wherein progenitor malignant hepatocytes appear and thrive in a complex cellular and cytokine milieu until gaining to exert a cancer surveillance function, primarily acting to suppress tumorigenesis (For a brief animation please visit https://www.youtube.com/watch?v=gGCjekCl5O4). We are now testing whether these aberrant immune foci could serve as new targets for cancer therapy.

Epithelial p53 and the microenvironment – a continuous cross talk

p53 is a central hub in preventing cancer: it is regulated by multiple cellular signaling pathways and biochemical events and in turn regulates the expression of multiple target genes and can execute several cellular outcomes. However, as cancer develops in tissues it is only logical to assume that p53 could be regulated by, and in turn regulate, tissue level phenomena which transcend the cellular level. Indeed, we have recently noted that p53 regulates the crosstalk between gut epithelial cells and the underlying lamina propria, to preserve tissue boundaries. We will explore the hypothesis that inflammation, injury or repair change the amplitude or the spectrum of the p53 response in epithelial cells. We expect that tissue danger lowers the threshold for p53 activation and thus adds extra-protection. Furthermore, by modulating the nature of the p53 response, the tissue could adapt better to the changing environment. We will study the reciprocal hypothesis that p53 in epithelial cells modulates the state of the microenvironment in disease states. Thus, it is conceivable that genotoxic stress occurring in multiple epithelial cells (leading to p53 activation), should alter inflammatory processes, to modulate inflammation so that the latter will not increase cancer risk.

Metabolic control of sperm lineage transitions

We have recently established a novel method for isolating germ cells from mouse testis. Our method utilizes transgenic mice that express tomato fluorescent protein under germ cell specific promoter allowing us to perform sorting of cells by fluorescence activated cell sorting. We are able to isolate 7 distinct populations along the complex process of spermatogenesis: undifferentiated spermatogonia, differentiating spermatogonia, late spermatogonia, leptotene/zygotene (L/Z), pachytene, secondary spermatocytes and round spermatids.

This allowed us to characterize transcriptional and metabolic profiles of different stages, which suggest key involvement of metabolic pathways in controlling phenotypic transitions, tightly linked to the meiotic process, and likely affecting the balance between stemness and differentiation.

Our aim is to decipher the contribution of metabolic process that are uniquely active in specific stages of this intricate cell lineage to differentiation decisions. In addition to revealing the basic mechanisms of spermatogenesis we hope that our findings could open new ways for combatting infertility as well as germ cell tumors.

1. Kanarek N, Grivennikov SI, Leshets M, Lasry A, Alkalay I, Horwitz E, Shaul YD, Stachler M, Voronov E, Apte RN, Pagano M, Pikarsky E, Karin M, Ghosh S, Ben-Neriah Y, Critical role for IL-1β in DNA damage-induced mucositis, Proc Natl Acad Sci U S A. 111:E702-11 (2014).
2. Shilo A, Ben Hur V, Denichenko P, Stein I, Pikarsky E, Rauch J, Kolch W, Zender L & Karni R, Splicing factor hnRNP A2 activates the Ras-MAPK-ERK pathway by controlling A-Raf splicing in hepatocellular carcinoma development. RNA. 20:505-15 (2014).
3. Horwitz E, Stein I, Andreozzi M, Nemeth J, Shoham A, Pappo O, Schweitzer N, Tornillo L, Kanarek N, Quagliata L, Zreik F, Porat RM, Finkelstein R, Reuter H, Koschny R, Ganten T, Mogler C, Shibolet O, Hess J, Breuhahn K, Grunewald M, Schirmacher P, Vogel A, Terracciano L, Angel P, Ben-Neriah Y & Pikarsky E. Human and mouse VEGFA-amplified hepatocellular carcinomas are highly sensitive to sorafenib treatment. Cancer Discovery. 4:730-43 (2014).
4. Yanger K*, Knigin D*, Zong Y,  Maggs L, Gu G, Akiyama H, Pikarsky E, and  Stanger BZ. Adult hepatocytes are formed by self-duplication rather than stem-cell differentiation, Cell Stem Cell. 15:340-9 (2014).
5. Hefetz-Sela S, Stein I, Klieger Y, Porat R, Sade-Feldman M, Zreik F, Nagler A, Pappo O, Eferl R, Wagner EF, Ben-Neriah Y, Baniyash M, and Pikarsky E. Acquisition of an Immunosuppressive Pro-Tumorigenic Macrophage Phenotype Depends on c-Jun Phosphorylation,  Proc Natl Acad Sci U S A. 111:17582-7 (2014).
6. Gluschnaider U, Hertz R, Ohayon S, Smeir S, Smets M, Pikarsky E, and Bar-Tana J. Long-Chain Fatty Acid Analogues Suppress Breast Tumorigenesis and Progression, Cancer Res. 74:6991-7002 (2014).
7. Kravtsova-Ivantsiv Y, Shomer I, Cohen-Kaplan V, Snijder B, Superti-Furga g, Gonen H, Sommer T, Ziv T, Admon A, Naroditsky I, Jbara M, Brik A, Normand R, Shen-Orr SS, Pikarsky E, Kwon YT, Doweck I, and Ciechanover A. KPC1-Mediated Ubiquitination and Proteasomal Processing of NF-κB1 p105 to p50 Restricts Tumor Growth. Cell. 161:333-47 (2015).
8. Gilad R, Meir K, Stein I, German L, Pikarsky E & Mabjeesh NJ. High SEPT9_i1 protein expression is associated with high-grade prostate cancers. PLoS ONE. 10:e0124251 (2015).
9. Abu-Remaileh M, Bender S, Raddatz G, Ansari I, Cohen D, Gutekunst J, Musch T, Linhart H, Breiling A, Pikarsky E, Bergman Y & Lyko F. Chronic inflammation induces a novel epigenetic program that is conserved in intestinal adenomas and in colorectal cancer. Cancer Res. 75:2120-30 (2015).
10. Falick Michaeli T, Laufer N, Sagiv JY, Dreazen A, Granot Z, Pikarsky E, Bergman Y & Gielchinsky Y. The rejuvenating effect of pregnancy on muscle regeneration. Aging Cell. 14:698-700 (2015).
11. Lazar CH, Kimchi A, Namburi P, Mutsuddi M, Zelinger L, Beryozkin A, Ben-Simhon S, Obolensky A, Ben-Neriah Z, Argov Z, Pikarsky E, Fellig Y, Marks-Ohana D, Ratnapriya R, Banin E, Sharon D & Swaroop A. Nonsyndromic Early-Onset Cone-Rod Dystrophy and Limb-Girdle Muscular Dystrophy in a Consanguineous Israeli Family are caused by Two Independent yet Linked Mutations in ALMS1 and DYSF. Hum Mutat. 36:836-41 (2015).
12. Finkin S, Yuan D, Stein I, Taniguchi K, Weber A, Unger K, Browning JL, Goossens N, Nakagawa S, Gunasekaran N, Schwartz ME, Kobayashi M, Kumada H, Berger M, Pappo O, Rajewsky K, Hoshida Y, Karin M, Heikenwalder M, Ben-Neriah Y & Pikarsky E. Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma. Nature Immunology, 16:1235-44 (2015).
13. Horwitz E, Stein I, Ben-Neriah Y & Pikarsky E. Animal model studies indicate a candidate biomarker for sorafenib treatment of hepatocellular carcinoma. Molecular & Cellular Oncology. 2:1, e968028 (2015).
14. Hefetz-Sela S, Stein I & Pikarsky E. Restoring inflammatory balance as a potential preventive strategy for inflammation induced cancer. Oncoimmunology. 4:e1039764 (2015).
15. Li D, Fu J, Du M, Zhang H, Li L, Cen J, Li W, Chen X, Lin Y, Conway EM, Pikarsky E, Wang H, Pan G, Ji Y, Wang HY, Hui L. Hepatology. 64(4):1105-20 (2016).
16. Darash-Yahana M, Pozniak Y, Lu M, Sohn YS, Karmi O, Tamir S, Bai F, Song L, Jennings PA, Pikarsky E, Geiger T, Onuchic JN, Mittler R, Nechushtai R. Breast cancer tumorigenicity is dependent on high expression levels of NAF-1 and the lability of its Fe-S clusters. Proc Natl Acad Sci USA. 113(39):10890-5 (2016).
17. Holt SH, Darash-Yahana M, Sohn YS, Song L, Karmi O, Tamir S, Michaeli D, Luo Y, Paddock ML, Jennings PA, Onuchic JN, Azad RK, Pikarsky E, Cabantchik IZ, Nechushtai R & Mittler R. Activation of apoptosis in NAF-1-deficient human epithelial breast cancer cells. J Cell Sci. 129:155-65 (2016).
18. Tarcic O, Pateras IS, Cooks T, Shema E, Kanterman J, Ashkenazi H, Boocholez H, Hubert A, Rotkopf R, Baniyash M, Pikarsky E, Gorgoulis VG & Oren M. RNF20 Links Histone H2B Ubiquitylation with Inflammation and Inflammation-Associated Cancer, Cell Rep., 14:1462-76 (2016).
19. Katlinskaya YV, Katlinski KV, Lasri A, Li N, Beiting DP, Durham AC, Yang T, Pikarsky E, Lengner CJ, Johnson FB, Ben-Neriah Y & Fuchs SY. Type I Interferons Control Proliferation and Function of the Intestinal Epithelium. Mol Cell Biol. 36:1124-35 (2016).
20. Samarin J, Laketa V, Malz M, Roessler S, Stein I, Horwitz E, Singer S, Dimou E, Cigliano A, Bissinger M, Falk CS, Chen X, Dooley S, Pikarsky E, Calvisi DF, Schultz C, Schirmacher P & Breuhahn K. PI3K/AKT/mTOR-dependent stabilization of oncogenic far-upstream element binding proteins in hepatocellular carcinoma cells, Hepatology. 63:813-26 (2016).
21. Llovet JM, Zucman-Rossi J, Pikarsky E, Sangro B, Schwartz M, Sherman M, Gores G. Hepatocellular carcinoma, Nature Reviews Disease Primers. 2:1-23 (2016).
22. Pikarsky E, Heikenwalder M. Focal and Local: Ectopic Lymphoid Structures and Aggregates of Myeloid and Other Immune Cells in Liver. Gastroenterology. 151:780-783. (2016).
23. Malakar P, Shilo A, Mogilevsky A, Stein I, Pikarsky E, Nevo Y, Benyamini H, Elgavish S, Zong X, Prasanth KV, Karni R.Long Noncoding RNA MALAT1 Promotes Hepatocellular Carcinoma Development by SRSF1 Upregulation and mTOR Activation. Cancer Res. 77:1155-1167. (2017).
24. Tarcic O, Granit RZ, Pateras IS, Masury H, Maly B, Zwang Y, Yarden Y, Gorgoulis VG, Pikarsky E, Ben-Porath I, Oren M. RNF20 and histone H2B ubiquitylation exert opposing effects in Basal-Like versus luminal breast cancer. Cell Death Differ. 24:694-704. (2017).
25. Etzioni A, Ciechanover A, Pikarsky E. Immune defects caused by mutations in the ubiquitin system. J Allergy Clin Immunol. 139:743-753. (2017).
26. Hanin G, Yayon N, Tzur Y, Haviv R, Bennett ER, Udi S, Krishnamoorthy YR, Kotsiliti E, Zangen R, Efron B, Tam J, Pappo O, Shteyer E, Pikarsky E, Heikenwalder M, Greenberg DS, Soreq H. miRNA-132 induces hepatic steatosis and hyperlipidaemia by synergistic multi-target suppression. Gut. 67:1124-1134 (2017).
27. Yuan D, Huang S, Berger E, Liu L, Gross N, Heinzmann F, Ringelhan M, Connor TO, Stadler M, Meister M, Weber J, Öllinger R, Simonavicius N, Reisinger F, Hartmann D, Meyer R, Reich M, Seehawer M, Leone V, Höchst B, Wohlleber D, Jörs S, Prinz M, Spalding D, Protzer U, Luedde T, Terracciano L, Matter M, Longerich T, Knolle P, Ried T, Keitel V, Geisler F, Unger K, Cinnamon E, Pikarsky E, Hüser N, Davis RJ, Tschaharganeh DF, Rad R, Weber A, Zender L, Haller D, Heikenwalder M. Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS. Cancer Cell. 31:771-789 (2017).
28. Heikenwälder M, Pikarsky E. Learning the Roles of the Hepatic Adaptive Immune System in Hepatocellular Carcinoma-Nature’s Guide for Successful Cancer Immunotherapy. Semin Liver Dis. 37(3):210-218 (2017).
29. Ringelhan M, Pfister D, O’Connor T, Pikarsky E, Heikenwalder M. The immunology of hepatocellular carcinoma. Nat Immunol. 19(3):222-232 (2018).

• Yinon Ben-Neriah, Hebrew University, Jerusalem, Israel
• Yehudit Bergman, Hebrew University, Jerusalem, Israel
• Aaron Ciechanover, Rappaport Faculty of Medicine and Research Institute, Haifa, Israel
• Mathias Heikenwalder, German Cancer Research Center (DKFZ), Heidelberg, Germany
• Shulamit Katzav, Hebrew University, Jerusalem, Israel
• Ana Martin-Villalba, German Cancer Research Center (DKFZ), Heidelberg, Germany
• Moshe Oren, Weizmann Institute of Science, Rehovot, Israel
• Oren Parnas, Hebrew University, Jerusalem, Israel
• Varda Rotter, Weizmann Institute of Science, Rehovot, Israel