Manolis C. Demetriou

The role of alpha 6 integrin in prostate cancer migration and bone pain in a novel xenograft model.

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain.

Integrin clipping: A novel adhesion switch?

During human prostate cancer progression, the majority of normally expressed integrins are suppressed with the exception of the α6, α3, and β1 integrins. We have shown that in prostate cancer, the α6 integrin is found paired with the β1 integrin and that a novel form of the α6 integrin that lacks a large portion of the extracellular domain (α6p) exists. The α6pβ1 integrin is found in human prostate cancer tissue specimens as well as tissue culture cell lines and is formed on the cell surface. This review discusses the mechanism of α6pβ1 production and the potential functions of this integrin variant. Our current working model predicts that the α6pβ1 integrin maintains the intracellular cytoskeletal connections associated with the heterodimer while allowing for an alteration in cell adhesion. The mechanism provides a selective advantage for cancer cell metastasis.

alpha6 Integrin Cleavage: Sensitizing human prostate cancer to ionizing radiation

Purpose: The goal was to determine if prostate tumor cells containing a mutant α6 integrin would be defective in tumor re-population following clinically relevant fractionated ionizing radiation (IR) treatments. Material and methods: Human prostate cancer cells derived from PC3N cells were used which conditionally expressed a cleavable, wild type form of α6 integrin (PC3N-α6-WT) or a mutated non-cleavable form of α6 integrin (PC3N-α6-RR). The resulting tumor growth before, during and after fractionated doses of IR (3 Gy×10 days) was analyzed using the endpoints of tumor growth inhibition (T/C), tumor growth delay (T-C), tumor doubling time (Td) and tumor cell kill (Log10 cell kill). Results: The T/C values were 36.1% and 39.5%, the T-C values were 20.5 days and 28.5 days and the Td values were 5.5 and 10.5 days for the irradiated PC3N-α6-WT and PC3N-α6-RR cells, respectively. The Log10 was 1.1 for the PC3N-α6-WT cells and 0.8 for the PC3N-α6-RR cells. The tumor response to IR was altered in tumors expressing the mutant α6 integrin as indicated by a significant increase in tumor growth inhibition, an increase in tumor growth delay, an increase in tumor doubling time and an increase in tumor cell kill. Conclusions: Blocking integrin cleavage in vivo may be efficacious for increasing the IR responsiveness of slow growing, pro-metastatic human prostate cancer.

Integrin A6 Cleavage in Mouse Skin Tumors

We have previously identified a structural variant of the α6 integrin (Laminin receptor) called α6p. The α6p variant is a 70 kDa form of the full-length α6 integrin (140 kDa) that remains paired with either the β1 or β4 subunit on the cell surface. α6p is produced by urokinase-type plasminogen activator (uPA), which removes the extracellular β-barrel domain while the receptor is on the cell surface. The α6p integrin was present in human prostate cancer tissue but not in normal tissue and the cleavage of the α6 integrin extracellular domain promotes tumor cell invasion and migration on laminin. The objective of the present study was to determine whether the α6p integrin is observed in other models of carcinogenesis. Our results indicate detectable low levels of α6p in normal mouse skin, and comparatively elevated levels in mouse papillomas and squamous cell carcinomas induced by DMBA, TPA and MNNG treatments. Furthermore, we have found that α6p was present at high levels in skin melanomas of transgenic mice that over express activated Ha-ras under the control of the tyrosinase promoter. Finally, subcutaneous injection into athymic nude mice of a malignant mouse keratinocyte derived cell line (6M90) that is α6p negative, results in the development of tumors that contain α6p integrin. The latter results indicate that α6p is induced in vivo suggesting that the tumor microenvironment plays a major role in the production of α6p. Taken together, these data suggest that the cell surface cleavage of the α6 integrin may be a novel mechanism of integrin regulation and might be an important step during skin tissue remodeling and during carcinogenesis.

A molecular approach for the diagnosis of wood rotting disease in desert citrus

Brown heartwood rot is an increasingly important problem in southwestern deserts, affecting numerous native tree species, and commercial lemon production. Two fungal pathogens, Coniophora ere- mophila and Antrodia sinuosa, have been identified as causal agents of brown heartwood rot in citrus. Development of effective disease management strat- egies relies on the ability to readily identify and dis- tinguish between the pathogens; therefore we have developed molecular markers as an alternative to morphometric methods to differentiate these two economically important pathogens. Random ampli- fied polymorphic DNA analysis identified four prim- ers that revealed fungal polymorphisms. Polymorphic DNA fragments were cloned, sequenced, and used to create a 700 bp sequence characterized amplified re- gion (SCAR) in C. eremophila isolates using sequence- specific primers, Conio7 and Conio8. Three frag- ments (300, 650, and 1000 bp) were amplified with Conio7 and Conio8 in A. sinuosa isolates. A rapid, small scale method for DNA extraction from fungus was developed to expedite the technique. With this method, the usefulness of the Conio7 and Conio8