Microbes and Cancer

1. Microbes and Cancer

2. Use of bacteria in the treatment cancer killed bacteria live bacteria bacterial toxins Association of bacteria with cancer Helicobacter pylori (induction) probiotics (inhibition)

3. Use of bacteria in the treatment of cancer

4. Imhotep (c 2600 BC) - an Egyptian physician - father of medicine “recommended treatment for tumors was poultice followed by incision” By 600 B.C., Imhotep--architecture and chief minister of King Djoser--had become a demigod.... Greece and Rome saw in him their god of healing until Christianity swept him away.” (www.cwo.com/~lucumi/ imhotep-tr.gif) History of cancer treatment Peregrine Laziosi (1265-1345) -patron saint of cancer afflicted with a malignant leg tumor / amputation recommended tumor grew until it broke through the skin / became infected “such a horrible stench was given off that it could be endured by no one sitting by him” by the time of surgery there were no signs of the tumor In M. Stanislas Tanchou (1844) - treatise on cancer ‘‘One knows that often the affected lymph nodes and primary growths disappear during the course of concurrent illness, never to return. It is according to that idea … that a large number of observers have advised establishing ‘issues’ [suppurating sores] on diverse portions of the body and even in the wounds remaining after operation’’.

5. (www.cancerdecisions.com) History - Microbial anti-tumor therapy “nature’s remedy for cancer” tumor regression followed by bacterial, fungal, viral or protozoal infections William Coley - (NY surgeon - late 1800s)- linked erysipelas infection following sarcoma operation to tumor remission • Infected next 10 patients with erysipelas (Streptococcus pyogenes) • Switched to killed bacterial treatment (S. pyogenes / Serratia marcescens) “Coley's toxins” • Used by others to treat carcinomas, lymphomas, melanomas, myelomas • - able to induce recoveries in final • stages of disease

6. Treatment with Coley’s toxins Requirements of an effective regimen • imitate a naturally occurring infection • essential to induce a fever injections administered daily or every other day dosage gradually increased with time (to avoid immune tolerance) vaccine injected directly into tumor (and metastases) followed by >6 mo course of weekly injections to prevent recurrence 1931 -in Modern Operative Surgery states “after amputation, prophylatic injections of Coley’s fluid should be given in doses sufficient to cause a sharp febrile response” 1936 - Coley’s vaccine endorsed in the New and Nonofficial Remedies of the American Medical Association “its use as a prophylactic in conjunction with conservative or radical surgery” and “inoperable cases may be quite justified”

7. (SAH Cann et al. Postgrad. Med. 79:672-680, 2003) Mechanism of tumor regression by Coley’s toxins Induces enhanced immune response to cancer tissue Active component - complex reflects immune response to pathogen multitude of cytokine cascades diverse cellular & humoral immune responses draws attention to innate immune response in early onset of tumor regression (treatments must be continued until complete regression of tumor) Immune system functions to defend against pathogens & in tissue repair Tumor - (partly self / partly foreign) - likened to a wound healing response (leukocyte mediated extracellular matrix degradation / growth factor production / induction of new blood and lymphatic vessels) Coley’s toxins - shift the immune response balance to defensive mode tumor vasculature more susceptible to fibrile immunostimulated collapse

8. Decline in use Coley’s toxins 1st -20th century - initiated use of sterile cancer surgical procedures 2nd -Coley’s death in 1936 - coincided with introduction of - radiotherapy cancer treatments & gaining of acceptance of chemotherapy (both treatments more easily standardized) but counter to immunotherapy - both highly immunosuppressive 3rd -following WWII - increased antibiotic use further decreased post- surgical infections / severity & duration of infections 4th -antipyretics came into routine use to eliminate discomforting symptoms of immune response result -reports of spontaneous tumor regression became less common Report in NEJM (1972) by Rukcdeschel et al. “Postoperative empyema improves surivial in lung cancer: documentation of a natural experiment”

9. Retrospective evaluation of Coley’s regimen In - comparison of 10 year survival rates of: past successes using Coley’s regimen to modern conventional cancer treatment (using data from Surveillance Epidemiology End Result cancer registry) “patients receiving modern conventional therapies did not fare better than patients receiving treatment initiated by Coley over 100 years ago” 1963 -US FDA assigned Coley’s vaccine a “new drug” status ruling forces expensive testing protocols - essentially bars use of Coley’s treatment on cancer patients -

10. Current studies using microbes to treat cancer

11. Bacterial toxins Live bacteria

12. Requirements of live bacterial anti-tumor agents • Selective toxicity to tumor cells • Infect and multiply within tumor • Function at low number • Sensitive to antibiotics

13. Live bacterial anti-tumor agents Bacillus Calmette-Guerin (BCG) Attenuated Mycobacterium bovis Anaerobic bacteria: Clostridium novyi / Clostridium sordellii spores Bifidobacteria spp. Clostridium producing / delivering anti-tumor agents Salmonella: Salmonella typhimurium Salmonella expressing pro-drug modification agents Pseudomonas: Genetically modified Pseudomonas aeruginosa

14. Extended disease-free survival seen with BCG treatment of bladder cancer BCG is the single most effective intravesical agent with complete response rates of ~55-75% for residual disease and CIS (cancer in situ), respectively. Two times more effective than chemotherapy for prophylaxis. Two-thirds of responses persist 5 years. BCG generally used more cautiously than chemotherapy due to low (<5%) but serious risk of systemic BCG infection (BCGosis) especially if administered in setting of recent surgery or traumatic catheterization. (Reproduced from Patard et al. Urology. 58:551-, 2001.) BCG - bacillus Calmette-Guerin vaccine only conventional bacterial treatment in use used to treat superficial bladder cancer applied directly to tumor site (intra-vesicle administration following surgery) repeated courses of administration induces complex immune response IL-1, IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, INFg, TNFa, MCSF

15. Distribution of anaerobic bacteria within tumors Mice bearing s.c. B16 tumors were injected i.v. with 5x107 live B. longum bacteria or wild-type C. novyi spores. Mice receiving B. longum were given i.p. injections of lactulose daily for 5 days to increase bacterial growth, then killed for analysis of tumor colonization. Mice receiving C. novyi spores were killed one day after injection for analysis. (A/B)B. longum experiment, showing low- and high-power views of the bacteria (stained deep blue) clustered within a colony. (C)C. novyi experiment, showing dispersion of bacteria throughout the necrotic region of the tumor. (D) High-power view, showing invasion of C. novyi bacteria into surrounding viable tumor cells (stained purple) on the Left. Gram-stain shows - B. longum concentrated within a few colonies C. novyi dispersed throughout the poorly vascularized portions of the tumors (Dang et al, PNAS, 98:15155-15160, 2001) Use of anaerobic bacteriain cancer treatment Solid tumors include large hypoxic, poorly vascularized regions limits effectiveness - ionizing radiation(dependent on oxygen) chemotherapy treatments(require vascularization) 1947-1980 - Recognized selective growth of anaerobic bacteria in hypoxic regions of solid tumors

16. Histologic examination of HCT116 xenografts before and after treatment a) Glut-1 staining (facilitative glucose transporter) of untreated HCT116 xenograft reveals islands of well oxygenated cells (O) interspersed within regions of hypoxia (H). In some cases, blood vessels could be observed in the middle of the islands (arrowheads in a and b). Necrotic regions (N) are poorly stained for Glut-1. b) H&E stain of a serial section of xenograft shown in a. c) H&E stain of HCT116 tumor xenograft 96 h after i.v. injection of spores, showing extensive central necrosis and remaining viable tumor rim (arrows). d) H&E stain of HCT116 tumor xenograft 96 h after i.v. injection of spores when given in combination with radiation. No viable tumor cells could be observed on several sections throughout the tumor. (Original magnification, x40.) (Bettegowda et al. PNAS, 100:15083-15088 , 2003) Use of Clostridium in cancer treatment 2001 -Bert Vogelstein -combination bacteriolytic therapy (COBALT) Clostridium novyi with chemo- or radiation therapy to treat HCT116 colon carcinoma / B16 melanoma cells implanted in mice - “new dimension”- in cancer treatment

17. Clostridium-directed enzyme pro-drug therapy (CDEPT) - cytosine deaminase (CD) + 5-fluorocytosine => 5-fluorouracil anti-tumor agent expression of CD by C. sporogenesin mice => significant anti-tumor effect (Michl and Gress, Current Cancer Drug Targets, 2004) Clostridium-directed cancer treatments Clostridium spore treatment alone -not sufficient for complete tumor regression Clostridium expression of (recA-mediated) irradiation-induced TNF-a resulted in a 242% increase in TNF-a secretion

18. * (Sznol, M. et al. J. Clin. Invest. 2000;105:1027-1030) *Attenuated by reduction in lipid A expression and deletion of the purI (purine) gene. Treatment of tumors with Salmonella 1997 -Pawlek et al reported IV administered Salmonella accumulated in implanted tumors in mice at levels >200-1000-fold that of normal tissue / retarded tumor growth

19. Treatment of tumor patients with Salmonella Phase I study:IV administration of attenuated S. typhimurium (strain VNP20009) to patients with metastatic melanoma(J Clin Oncol. 20:142-152, 2001) • Results: • Maximum tolerated dose - 3x108 cfu/m2 (x body surface area) • dose limiting toxicity -thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, hypophosphatemia • VNP20009 induced dose related increase in proinflammatory cytokines • IL-1b, TNFa, IL-6, IL-12 • VNP20009 - rapidly cleared from bloodstream (< 4h) • Focal tumor colonization - observed in 2 patients • None of the patients experienced objective tumor regression • Conclusion: • VNP20009 can be administered safely to patients • Some tumor colonization was observed • No anti-tumor effects were observed Reason for differences in tumor accumulation / colonization of rodent models vs. human human remains unknown

20. Salmonella anti-tumor activity Mechanism for Salmonella anti-tumor activity in rodents: Low dose IV injected Salmonella initially accumulates in spleen and liver By 24 h Salmonella detected in other tissues (with higher concentrations in tumors) Accumulation and reduction in tumors may relate to: - induction of inflammatory response - type III secretion of toxins - vasculature (or trapping) of bacteria in tumors - delivery to tumors via macrophages - favored growth in nutrient-rich / hypoxic / necrotic areas of tumors - protected tumor environment (devoid of granulocytes / antibodies Alternative strategies for Salmonella anti-tumor treatment of patients: Use for delivery of therapeutic pro-drugs (via intra-tumoral injection) - cytosine deaminase (TAPET-CD) + 5-FC => 5-FU accumulation in tumors In two patients TAPET-CD Salmonella treatment found to convert 5-FC => 5-FU with no systemic spread of bacteria Identifies the potential use of Salmonella as a pro-drug tumor delivery agent

21. Relates to type III secretory process of P. aeruginosa ExoS ExoT ExoU ExoY ExoS Translocation Pa. Use of Pseudomonas aeruginosa in tumor therapy

22. Increased sensitivity of tumor cells to P. aeruginosa T3S - ExoS 388DS 388 [3H]Thymidine incorporation (% of control) Normal epithelial Tumor derived Fibroblasts Myeloid Cell Lines

23. Pseudomonas-tumor cell interaction model T3S ExoS T3S ExoS T3S ExoS

24. Bacterial toxin anti-tumor agents Diphtheria toxin (DT) Pseudomonas exotoxin A (PE) Clostridium perfringes type A enterotoxin (CPE)

25. Diphtheria toxin ADP-ribosyltransferase activity CONH2 EF2 N O O CH2 CH2 DT/PE CONH2 (Choe et al. 1992) + P P P P Pseudomonas exotoxin A N O Adenine O Adenine CH2 CH2 nicotinamide NAD ADP-ribosylated protein (Kabat, Iglewski, et al.) (Allured, et al, 1986) Toxic mechanism of diphtheria toxin and Pseudomonas exotoxin A

26. S S A-subunit B-subunit L enzyme activity /internalization / receptor binding intracellular targeting S S A-subunit B-subunit L enzyme activity /internalization / alter receptor binding alter intracellular targeting Design of toxins as anti-cancer agents (immunotoxins) Tumor cell cytotoxicity

27. (Michl and Gress, Current Cancer Drug Targets, 4:689-702, 2004) Diphtheria toxin (DT) preclinical studies

28. Results of DT preclinical & clinical studies Fusion immunotoxin constructs - cytotoxic in vitro: Tf-CRM107 - showed specificity for glioma cells DAB389EGF - specifically targeted pancreatic or breast cancer cells DT389HGR- shows specificity for Erb2/Her2 expressing breast cancer cells DT-VEGF - resulted in decreased tumor volume Clinical trials: Tf-CRM107 - intratumoral injection of patients with advanced solid brain tumors Observed - 50% reduction in tumor volume in 9 of 15 patients DAB389EGF - administered IV to metastatic carcinoma patients Limiting side effect - transient transaminasemia (toxicity to normal hepatocytes) Problems: Inconsistencies in toxicity - due to ligand folding Non-tumor associated toxicity - due to non-tumor cell receptor expression

29. (Michl and Gress, Current Cancer Drug Targets, 4:689-702, 2004) Pseudmonas exotoxin A (PE) clinical studies (1996-2003) LMB-1 -IV treatment of solid tumors expressing Le(Y) antigens - showed anti-tumor activity / main side effect vascular leakage syndrome scFvPPE40 -IV (fused variable chain of Ig to PE38) - smaller molecular weight / shorter half-life - less side effects IL-4-PE38-KDEL -intra-tumoral injection into glioblastomas - glioma necrosis in 6 of 9 patients / lacked signs of toxicity IL-13-PE38QQR -in phase I/II trials - intra-tumoral administration by convection enhanced delivery (CED) to adults with malignant glioma - studies indicate CED generally well tolerated / main side effect - grade IV fatigue 3 of 15 patients responded to treatment

30. (Miyakawa et al, Infect. Immun 73:8407-8410, 2005) Clostridium perfringes type A enterotoxin (CPE) toxin linked to C. perfringes food poisoning common human food-borne poisoning cause of antibiotic-associated diarrhea

31. (Michl and Gress, Current Cancer Drug Targets, 4:689-702, 2004) Toxic mechanism of C. perfringes enterotoxin CPE receptor -member of claudin multi-gene family major constituent of tight junctions - important in cell polarity & intercellular barriers claudin 4 - overexpressed in pancreatic /colon / breast / gastric cancers (as compared to normal tissue) In vitro CPE treatment => tumor necrosis (potential intra-tumoral injection of CPE to treat solid tumors or drug delivery to claudin-4 overexpressing tumor cells)

32. Association of bacterial infections with induction of cancer

33. Microbes associated with induction of cancer Peyton Rous (1911) - demonstrated a cell-free extract could transfer chicken malignancy - led to discovery of RNA retrovirus and src transforming gene - oncogene theory - Other viruses linked to cancer - papilloma virus - cervical cancer hepatitis viruses B and C - liver cancer Epstein-Barr virus -lymphoproliferative disorders / nasopharyngeal carcinoma / Burkitt lymphoma Parasitic infections - Schistosoma haematobium - bladder cancer Bacterial infections - Helicobacter pylori - gastric cancer Chlamydia psittaci - ocular lymphomas Campylobacter jejuni - small intestine lymphomas Salmonella typhi - hepatobiliary carcinoma

34. Characteristics of infections associated with cancer • Chronic infections • Associated with chronic inflammation • Afflict organ where cancer develops • Lack induction of acute febrile response • Generally reflects a failed immune response

35. Carcinogenic process Cancer arises when a cell acquires sufficient mutations to survive and multiply independently of its normal regulation by soluble extracellular factors and interaction with its neighbors (AJ Lax, Nature Reviews Microbiology, 2005) Bacterial infections: induce inflammation direct DNA damage cause immunosuppression increase angiogenesis affect cell signaling - proliferation inhibit cell cycle progression & apoptosis

36. Bacterial toxins with carcinogenic potential E. coli P. multocida E. coli H. pylori cytolethal distending toxin H. pylori (AJ Lax, Nature Reviews Microbiology, 2005)

37. 1983 -Barry Marshall, J. Robin Warren isolated H. pylori from human stomach biopsies First -identified as an infectious cause of peptic ulcer disease Later -H. pylori infection linked with increased risk of gastric adenocarcinoma & MALT (mucosa-associated lymphoid tissue) lymphoma 2005 -Barry J. Marshall and J. Robin Warren - awarded the Nobel Prize in Medicine or Physiology for “their discovery of the bacterium H. pylori and its role in gastritis and peptic ulcer disease" • Identifying the role of Helicobacter pylori in disease 1983 rejection letter describing their finding: “Dear Dr. Marshall, I regret that your research paper was not accepted for presentation. The number of abstracts that we receive continues to increase. For this meeting 67 were submitted and we could only accept 56.”

38. Identifying the role of Helicobacter pylori in disease To demonstrate that H. pylori was a pathogen needed to show: an association with disease evidence for immune reaction improved clinical outcome after eradication absence of beneficial effect of bacterium healthy people did not have H. pylori (most people with H. pylori are clinically well) Difficulty in fulfilling Koch’s postulates of H. pylori being a cause of cancer since cancer induction is multifactoral - relates to environmental factors, host factors and time Evidence: Pathogenesis research - H. pylori associated with gastric tumors Animal model studies -showed H. pylori infection induced gastric carcinoma Human epidemiology studies -identified a correlation between H. pylori infection and incidence of intestinal- and diffuse-type gastric carcinoma H. pylori - perturbs cell signaling mechanisms associated with cancer induction 1994 -H. pylori - declared a class 1 carcinogen

39. (www.bact.wisc.edu/ Bact330/bjmrjw.jpeg) (www.shef.ac.uk/.../ molmicro/hpylr110.html) • Helicobacter pylori Bacteriology: -Gram-negative, spiral shaped, microaerophilic bacterium (originally named Campylobacter pyloridis) - intimate / exclusive association with humans - co-existed with humans for much of human history - once believed to be a commensal - on-going evolution / diversification of H. pylori strains within a single host (>20% variation in genome content) - one-half of world’s population infected with H. pylori (highest rates in developing countries)

40. Colonization of the gastric mucosa by H. pylori (Giemsa stain) Gastrointestinal bleeding and erosive gastritis in association with H. pylori infection (www.bact.wisc.edu/ Bact330/lecturehelico2) • Helicobacter pylori • Disease: • -most common cause of gastritis in humans • (infiltration of the tissue with lymphocytes / • plasma cells) • - promotes peptic or gastric ulceration (10%) • - associated with mucosa-associated • lymphoid tissue lymphoma • -linked to gastric cancer (1%) • H. pylori protects against gastro-oesophageal influx and oesophageal carcinoma

41. (Willhite et al., J Biol Chem. 278:48204-48209, 2003) (Bauer et al., Infect Immun. 73::4643-4652, 2005) • Helicobacter pylori Virulence factors: - flagella - urease(stomach acid neutralization) - VacA -forms higher order structure intracellular membrane ion channels in vesicles & mitochondria - can induce apoptosis (found in all strains) - cag-cytotoxin associatedPAI (40 kb) encodes type IV secretion system induces pro-inflammatory cytokines (IL-8) (peptidoglycan dependent) - CagA - only protein substrate of cag PAI causes cell elongation “hummingbird phenotype” & cell scattering (enriched in strains from patients with severe disease)

42. Cag PAI type IV secretion A. Hypothetical configuration of the cag PAI T4SS based on Agrobacterium tumefaciens. B. Graphical representation of cag PAI form H. pylori strain 26695 showing relative gene size and orientation. CagA delivery and IL-8 induction phenotype of isogenic mutants are summarized below each gene from Fischer et al. 2001. (Bourzac and Guillemin, Cell Microbiol. 7:911-919, 2005)

43. CagA function * * carcinogenesis

44. Induction of malignancy by H. pylori Long term colonization / Chronic inflammation Stimulation of pro-inflammatory cytokines: IL8 - interleukin 8 COX2 - cyclooxygenase-2 ROS - reactive oxygen species NO - nitric oxide Tissue damage / Stimulation of signaling pathways: MAP - mitogen activaed protein kinases EGFR - epidermal growth factor receptor c-Met receptor kinase cAMP - elevated cyclic AMP Rap1 - Ras-related G-protein cell cycle deregulation Apoptosis - decrease / increase

45. goal - persistance co-evolves within host Development of H. pylori chronic infection homeostasis adapts to changing environment high mutation rate many bacterial populations exist H. pylori indigenous biota of human stomachs (natural habitat) chronic infection chronic inflammation induction of cancer

46. Concepts - Microbes & Cancer • Use of bacteria as an innate immune stimulus to treat cancer • Characteristics and approaches of using live bacterial anti-tumor agents • Characteristics and problems associated with the use of immunotoxin or bacterial toxin anti-tumor agents • Characteristics of bacterial infections associated with cancer induction

47. Important concepts in microbial pathogenesis • Abundance / diversity / distribution of bacteria in nature • Co-evolution / co-manipulation of prokaryotic & eukaryotic cells • Pathogenesis / virulence factors / experimental approaches to study bacterial pathogenesis • Bacterial toxin mechanisms - alternative uses of bacterial toxins • Strategies of intracellular pathogens • Bacterial secretion - role in virulence • Origin / strategies of emerging pathogens • Treatment and induction of cancer by bacteria • Host immune protection against pathogens