Microbes and Cancer

Use of bacteria in the treatment cancerkilled bacterialive bacteria

bacterial toxins

Association of bacteria with cancerHelicobacter pylori (induction)

probiotics (inhibition)

Use of bacteria in the treatment of cancer

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’’.

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)

Imhotep (c 2600 BC) - an Egyptian physician - father of medicine“recommended treatment for tumors was poultice followed

by incision”

History of cancer treatment

Peregrine Laziosi (1265-1345) - patron saint of cancer

afflicted with a malignant leg tumor / amputation recommendedtumor 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

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

(www.cancerdecisions.com)

Used by others to treat carcinomas, lymphomas, melanomas, myelomas

- able to induce recoveries in final stages of disease

Infected next 10 patients with erysipelas (Streptococcus pyogenes)

Switched to killed bacterial treatment (S. pyogenes / Serratia marcescens) “Coley's toxins”

Requirements of an effective regimen

imitate a naturally occurring infection essential to induce a fever

Treatment with Coley’s toxins

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”

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”

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

Induces enhanced immune response to cancer tissue

Mechanism of tumor regression by Coley’s toxins

Active component - complex

reflects immune response to pathogenmultitude 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 modetumor vasculature more susceptible to fibrile immunostimulated collapse

(SAH Cann et al. Postgrad. Med. 79:672-680, 2003)

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

Report in NEJM (1972) by Rukcdeschel et al. “Postoperative empyema improves surivial in lung cancer: documentation of a natural experiment”

result - reports of spontaneous tumor regression became less common

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 -

Current studies using microbes to treat cancer

Live bacteria

Bacterial toxins

Requirements of live bacterial anti-tumor agents

Selective toxicity to tumor cells Infect and multiply within tumor Function at low number Sensitive to antibiotics

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

used to treat superficial bladder cancerapplied directly to tumor site

(intra-vesicle administration following surgery)repeated courses of administrationinduces complex immune response

IL-1, IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, INF, TNF, MCSF

BCG - bacillus Calmette-Guerin vaccine only conventional bacterial treatment in use

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.)

Solid tumors include large hypoxic, poorly vascularized regions

limits effectiveness - ionizing radiation (dependent on oxygen)chemotherapy treatments (require vascularization)

Use of anaerobic bacteria in cancer treatment

1947-1980 - Recognized selective growth of anaerobic bacteria in hypoxic regions of solid tumors

(Dang et al, PNAS, 98:15155-15160, 2001)

Distribution of anaerobic bacteria within tumorsMice 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

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

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)

Clostridium-directed cancer treatments

Clostridium spore treatment alone - not sufficient for complete tumor regression

Clostridium expression of (recA-mediated) irradiation-induced TNF- resulted in a 242% increase in TNF- secretion

(Michl and Gress, Current Cancer Drug Targets, 2004)

Clostridium-directed enzyme pro-drug therapy (CDEPT) - cytosine deaminase (CD) + 5-fluorocytosine => 5-fluorouracil anti-

tumor agentexpression of CD by C. sporogenes in mice => significant anti-tumor effect

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

(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.

Phase I study: IV administration of attenuated S. typhimurium (strain VNP20009) to patients with metastatic melanoma (J Clin Oncol. 20:142-152, 2001)

Treatment of tumor patients with Salmonella

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-1, TNF, 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

Salmonella anti-tumor activity

Mechanism for Salmonella anti-tumor activity in rodents: Low dose IV injected Salmonella initially accumulates in spleen and liverBy 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

Use of Pseudomonas aeruginosa in tumor therapy

ExoS ExoT ExoU ExoY

Pa.

Relates to type III secretory process of P. aeruginosa

Translocation

ExoS

0

25

50

75

100

NK77 NK97 HT29 KB LNC DU HCT T24 SW SK/SH PA1 NIH Swiss HL60

Cell Lines

Normal epithelial Tumor derived Fibroblasts Myeloid

[3 H]T

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corp

ora

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% o

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388S388

Increased sensitivity of tumor cells to P. aeruginosa T3S - ExoS

Pseudomonas-tumor cell interaction model

T3SExoS

T3SExoS

T3SExoS

Bacterial toxin anti-tumor agents

Diphtheria toxin (DT)Pseudomonas exotoxin A (PE)

Clostridium perfringes type A enterotoxin (CPE)

Toxic mechanism of diphtheria toxin and Pseudomonas exotoxin A

(Kabat, Iglewski, et al.)

O

N

CH2

CH2

PP

Adenine

CONH2

DT/PE

EF2

+

ADP-ribosylated protein

nicotinamideO

OCH2

CH2

PP

AdenineON

CONH2

NAD

ADP-ribosyltransferase activity

Diphtheria toxin

(Choe et al. 1992)

Pseudomonas exotoxin A

(Allured, et al, 1986)

Design of toxins as anti-cancer agents

S SA-subunit B-subunit

L enzyme activity / internalization / alter receptor binding alter intracellular targeting

Tumor cell cytotoxicity

S SA-subunit B-subunit

L enzyme activity / internalization / receptor binding intracellular targeting

(immunotoxins)

(Michl and Gress, Current Cancer Drug Targets, 4:689-702, 2004)

Diphtheria toxin (DT) preclinical studies

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 cellsDT389HGR - shows specificity for Erb2/Her2 expressing breast cancer cellsDT-VEGF - resulted in decreased tumor volume

Problems:Inconsistencies in toxicity - due to ligand foldingNon-tumor associated toxicity - due to non-tumor cell receptor expression

Clinical trials:Tf-CRM107 - intratumoral injection of patients with advanced solid brain tumorsObserved - 50% reduction in tumor volume in 9 of 15 patients

DAB389EGF - administered IV to metastatic carcinoma patientsLimiting side effect - transient transaminasemia (toxicity to normal hepatocytes)

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

(Michl and Gress, Current Cancer Drug Targets, 4:689-702, 2004)

Pseudmonas exotoxin A (PE) clinical studies (1996-2003)

Clostridium perfringes type A enterotoxin (CPE)

toxin linked to C. perfringes food poisoningcommon human food-borne poisoningcause of antibiotic-associated diarrhea

(Miyakawa et al, Infect. Immun 73:8407-8410, 2005)

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)

(Michl and Gress, Current Cancer Drug Targets, 4:689-702, 2004)

Association of bacterial infections 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 cancerhepatitis 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 cancerChlamydia psittaci - ocular lymphomasCampylobacter jejuni - small intestine lymphomasSalmonella typhi - hepatobiliary carcinoma

Microbes associated with induction of cancer

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

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 inflammationdirect DNA damage

cause immunosuppressionincrease angiogenesis

affect cell signaling - proliferationinhibit cell cycle progression & apoptosis

Bacterial toxins with carcinogenic potential

(AJ Lax, Nature Reviews Microbiology, 2005)

E. coli

H. pylori

E. coli

P. multocida

H. pyloricytolethaldistending

toxin

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.”

Difficulty in fulfilling Koch’s postulates of H. pylori being a cause of cancer sincecancer induction is multifactoral - relates to environmental factors, host factors and time

To demonstrate that H. pylori was a pathogen needed to show:

an association with diseaseevidence for immune reaction

improved clinical outcome after eradicationabsence of beneficial effect of bacterium

healthy people did not have H. pylori

(most people with H. pylori are clinically well)

Identifying the role of Helicobacter pylori in disease

Evidence:

Pathogenesis research - H. pylori associated with gastric tumorsAnimal 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

Helicobacter pylori

(www.shef.ac.uk/.../ molmicro/hpylr110.html)

(www.bact.wisc.edu/ Bact330/bjmrjw.jpeg)

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)

Helicobacter pylori

Gastrointestinal bleeding and erosive gastritis in association with H. pylori infection

(www.bact.wisc.edu/ Bact330/lecturehelico2)

Colonization of the gastric mucosa by H. pylori (Giemsa stain)

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

Helicobacter pylori

(Willhite et al., J Biol Chem. 278:48204-48209, 2003)

(Bauer et al., Infect Immun. 73::4643-4652, 2005)

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 associated PAI (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)

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)

**

carcinogenesis

CagA function

Induction of malignancy by H. pylori

Tissue damage / Stimulation of signaling pathways:MAP - mitogen activaed protein kinasesEGFR - epidermal growth factor receptorc-Met receptor kinasecAMP - elevated cyclic AMPRap1 - Ras-related G-proteincell cycle deregulationApoptosis - decrease / increase

Long term colonization / Chronic inflammation Stimulation of pro-inflammatory cytokines:

IL8 - interleukin 8 COX2 - cyclooxygenase-2 ROS - reactive oxygen species NO - nitric oxide

Development of H. pylori chronic infection

H. pyloriindigenous biota of human stomachs

(natural habitat)

homeostasis co-evolves within host

goal -persistance

chronic infectionchronic inflammationinduction of cancer

adapts to changing environmenthigh mutation rate

many bacterial populations exist

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

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