Overexpression of claudin-3 and claudin-4 receptors in uterine serous papillary carcinoma: novel targets for a type-specific therapy using Clostridium perfringens enterotoxin (CPE).
BACKGROUND
Uterine serous papillary carcinoma (USPC) represents a highly aggressive variant of endometrial cancer. Using gene expression profiling, we recently identified high expression of the claudin-3 and claudin-4 receptors in a limited set of USPC. These tight junction proteins represent the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE) and are sufficient to mediate CPE binding and trigger subsequent toxin-mediated cytolysis. The potential for targeting this pathway in the treatment of USPC was explored.


METHODS
Claudin-3 and claudin-4 receptor expression was analyzed at the mRNA and protein levels in flash-frozen and formalin-fixed, paraffin-embedded tissue from 20 consecutive USPC patients. The potential of recombinant CPE as a novel therapy against primary, metastatic, and chemotherapy-resistant USPC cell lines was also investigated in vitro. Finally, the in vivo therapeutic effect of sublethal doses of CPE was studied in SCID mouse xenografts harboring subcutaneous and intraperitoneal USPC that expressed claudin-3 and claudin-4.


RESULTS
In all, 100% (20 out of 20) of the primary flash-frozen USPC tested overexpressed 1 or both CPE receptors by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Membranous immunoreactivity for claudin-4 protein expression was documented in the majority of USPC specimens tested by immunohistochemistry, whereas only a low level of membranous staining was found in normal endometrial control tissue samples. When primary and metastatic short-term USPC cell lines were incubated with different concentrations of CPE in vitro, a dose-dependent cytotoxic effect was demonstrated. In vivo, intratumoral injections of well-tolerated doses of CPE in large subcutaneous USPC xenografts led to large areas of tumor cell necrosis and tumor disappearance in all the treated animals, whereas sublethal intraperitoneal injections of CPE had a significant inhibitory effect on tumor progression, with extended survival of animals harboring chemotherapy-resistant intra-abdominal USPC carcinomatosis.


CONCLUSIONS
Claudin-3 and claudin-4 receptors may offer promising targets for the use of CPE as a novel type-specific therapy against this highly aggressive and chemotherapy-resistant variant of endometrial cancer.
Santin
Alessandro D
AD
Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA. santinalessandrod@uams.edu
Bellone
Stefania
S
Marizzoni
Moira
M
Palmieri
Michela
M
Siegel
Eric R
ER
McKenney
Jesse K
JK
Hennings
Leah
L
Comper
Fabrizio
F
Bandiera
Elisabetta
E
Pecorelli
Sergio
S
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D062465:Claudin-3; D062506:Claudin-4; D003016:Clostridium perfringens; D018283:Cystadenocarcinoma, Papillary / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D018284:Cystadenocarcinoma, Serous / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D019008:Drug Resistance, Neoplasm; D004768:Enterotoxins / Q000627:therapeutic use*; D005260:Female; D015870:Gene Expression; D006801:Humans; D007274:Injections, Intraperitoneal; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
C568122:CLDN3 protein, human; C568157:CLDN4 protein, human; D062465:Claudin-3; D062506:Claudin-4; C568123:Cldn3 protein, mouse; C568158:Cldn4 protein, mouse; D004768:Enterotoxins; D008565:Membrane Proteins; D012333:RNA, Messenger; C035598:enterotoxin, Clostridium