Lack of radiation dose or quality dependence of epithelial-to-mesenchymal transition (EMT) mediated by transforming growth factor ß.

PURPOSE: Epithelial-to-mesenchymal transition (EMT) is a phenotype that alters cell morphology, disrupts morphogenesis, and increases motility. Our prior studies have shown that the progeny of human mammary epithelial cells (HMECs) irradiated with 2 Gy undergoes transforming growth factor ß (TGF-ß)-mediated EMT. In this study we determined whether radiation dose or quality affected TGF-ß-mediated EMT. METHODS AND MATERIALS: HMECs were cultured on tissue culture plastic or in Matrigel (BD Biosciences, San Jose, CA) and exposed to low or high linear energy transfer (LET) and TGF-ß (400 pg/mL). Image analysis was used to measure membrane-associated E-cadherin, a marker of functional epithelia, or fibronectin, a product of mesenchymal cells, as a function of radiation dose and quality. RESULTS: E-cadherin was reduced in TGF-ß-treated cells irradiated with low-LET radiation doses between 0.03 and 2 Gy compared with untreated, unirradiated cells or TGF-ß treatment alone. The radiation quality dependence of TGF-ß-mediated EMT was determined by use of 1 GeV/amu (gigaelectron volt/atomic mass unit) (56)Fe ion particles at the National Aeronautics and Space Administration's Space Radiation Laboratory. On the basis of the relative biological effectiveness of 2 for (56)Fe ion particles' clonogenic survival, TGF-ß-treated HMECs were irradiated with equitoxic 1-Gy (56)Fe ion or 2-Gy (137)Cs radiation in monolayer. Furthermore, TGF-ß-treated HMECs irradiated with either high- or low-LET radiation exhibited similar loss of E-cadherin and gain of fibronectin and resulted in similar large, poorly organized colonies when embedded in Matrigel. Moreover, the progeny of HMECs exposed to different fluences of (56)Fe ion underwent TGF-ß-mediated EMT even when only one-third of the cells were directly traversed by the particle. CONCLUSIONS: Thus TGF-ß-mediated EMT, like other non-targeted radiation effects, is neither radiation dose nor quality dependent at the doses examined.

Integrin agonists as adjuvants in chemotherapy for melanoma.

PURPOSE: Metastatic melanomas are generally resistant to chemotherapy and radiation, even when wild-type for p53. These tumors often grow in small nests where many of the cells have little contact with extracellular matrix (ECM). Previous work showed that M21 melanomas undergo apoptosis in response to chemotherapy when cells are adherent to ECM but not in suspension. Thus, reduced integrin-dependent adhesion to ECM could mediate therapy resistance. The goal of this study was to test whether stimulation of integrin signaling could increase chemotherapeutic efficacy. EXPERIMENTAL DESIGN: Colony forming assays and survival assays were used to test the responses of melanoma lines in vitro. Severe combined immunodeficient mice with subcutaneous human melanomas received chemotherapy with or without reagents that stimulate integrin signaling; tumor volume was then monitored over time. RESULTS: Clonal growth assays confirmed that M21 cells showed reduced sensitivity to the chemotherapeutic drug 1-beta-D-arabinofuranosylcytosine (araC). When five additional primary melanoma lines were screened, 80% showed higher sensitivity when adherent compared with suspended. Subcutaneous M21 tumors in vivo showed minimal ECM between tumor cells. To evaluate the importance of integrin signaling in chemoresistance in this model, mice were treated with araC, with or without the multivalent snake venom disintegrin contortrostatin or the activating anti-beta1 integrin antibody TS2/16. Although araC, TS2/16, or contortrostatin alone had little effect on M21 tumor growth, combining araC with either integrin signaling reagents strongly reduced growth (P = 0001). CONCLUSIONS: Loss of integrin-mediated adhesion is rate limiting for therapeutic response in this model. Combining chemotherapy with reagents that stimulate integrin signaling may therefore provide a new approach to therapy.

New rationales for using TGFbeta inhibitors in radiotherapy.

PURPOSE: The first reports that ionizing radiation (IR) induces rapid and persistent activation of transforming growth factor beta1 (TGFbeta) were nearly two decades ago. Subsequent studies have shown that TGFbeta is a major mediator of cellular and tissue responses to IR and have revealed novel facets of its complex biology. RESULTS: We and others have recently shown that inhibition of production or signaling of TGFbeta in epithelial cells modulates radiosensitivity and impedes activation of the DNA damage response program. The primary transducer of cellular response to DNA damage caused by ionizing radiation is the nuclear protein kinase ataxia telangiectasia mutated, whose activity is severely compromised when TGFbeta is inhibited. Thus, in conjunction, with its well-recognized contribution to normal tissue fibrosis, the role of TGFbeta in the genotoxic stress program provides a previously unsuspected avenue to modulate radiotherapy. CONCLUSIONS: We hypothesize that identification of the circumstances and tumors in which TGFbeta manipulation enhances tumor cell radiosensitivity, while protecting normal tissues, could significantly increase therapeutic index.

Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition.

Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

1,25(OH)2D3 inhibits in vitro and in vivo intracellular growth of apicomplexan parasite Toxoplasma gondii.

The hormonal form of vitamin D, 1,25-dyhydroxyvitamin D3 (1,25(OH)2D3), is implicated in a wide range of functions other than its classical role in calcium and phosphorous homeostasis. When Toxoplasma gondii-infected BALB/c mice were treated with 1,25(OH)2D3, they succumb to death sooner than their counterparts. But they showed less parasite burden in tissues which was further supported by mild pathological lesions. As an effort to understand the physiological mechanism for the above observation an in vitro study was performed. Fewer parasites were observed when 1,25(OH)2D3 pre-treated murine intestinal epithelial cells were challenged with parasites. Moreover, the observed inhibition was dose-dependent and had a maximum effect with 10(-7)M of 1,25(OH)2D3. However, no observable difference was observed, when pre-incubated parasites were added to cells suggesting that the observed inhibition was a result of an effect from 1,25(OH)2D3 on Toxoplasma intracellular growth. Our data support the notion that 1,25(OH)2D3 may inhibit intra cellular T. gondii parasite proliferation in vivo and in vitro.

Stromelysin-3 is a potent negative regulator of adipogenesis participating to cancer cell-adipocyte interaction/crosstalk at the tumor invasive front.

The initial invasive processes during cancer development remain largely unknown. Stromelysin-3/matrix metalloproteinase 11 (ST3/MMP11) is associated with tumor invasion and poor prognosis. We present novel evidence that adipocytes present at human breast tumor invasive front are induced by cancer cells to express ST3. Using mouse syngeneic model, light and electron microscopy showed that in ST3-deficient mice but not in wild-type mice, forced cancer cell-adipocyte interaction/crosstalk results in adipocyte membrane alteration, allowing cancer cell fat infiltration and death. Thus, adipocytes are involved in initial cancer cell survival into connective tissue, and this effect is ST3 mediated. This suggested that ST3 might play a role in adipocyte metabolism. Accordingly, ST3-deficient mice exhibited fat excess and increased mRNA levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and adipocyte protein 2 (aP2) adipogenic markers, indicating that, in vivo, ST3 negatively regulates fat homeostasis. Moreover, ST3-deficient mouse embryonic fibroblasts exhibited a dramatic enhanced potential to differentiate into adipocytes associated with increased PPARgamma and aP2 expression, and recombinant ST3 treatment reverted their differentiation. Thus, in vitro, ST3 reduces adipocyte differentiation in an autocrine manner. High fibroblasts/adipocytes ratio is a stroma feature, and peritumoral fibroblast origin remains debated. Our results support the concept that invading cancer cells aberrantly restore the negative ST3 function on adipogenesis into proximal adipocytes/preadipocytes, leading to the accumulation/maintenance of a particular peritumoral fibroblast subpopulation. Accordingly, in human breast tumors, we observed that ST3-expressing peritumoral fibroblasts are distinct from alpha-smooth muscle actin-expressing myofibroblasts. This constitutes the first report of implication of a MMP in cancer cell-adipocyte interaction/crosstalk during early steps of connective tissue invasion.

Dosing and scheduling influence the antitumor efficacy of a phosphinic peptide inhibitor of matrix metalloproteinases.

The in vivo disposition and antitumor efficacy of a newly developed phosphinic matrix metalloproteinase inhibitor (RXP03) were examined. RXP03 potently inhibits MMP-11, MMP-8 and MMP-13, but not MMP-1 and MMP-7. Twenty-four hours after i.p. injection into mice, most of the RXP03 was recovered intact in plasma, feces (biliary excretion) and tumor tissue. Pharmacokinetic parameters indicated that, after an i.p. dose of 100 microg/day, the plasma concentration of RXP03 over 24 hr remained higher than the Ki values determined for MMP-11, MMP-8 and MMP-13. Efficacy of RXP03 on the growth of primary tumors induced by s.c. injection of C(26) colon carcinoma cells in mice was observed to depend both on RXP03 doses and treatment schedules. Tumor volumes in mice treated for 18 days with 50, 100 and 150 microg/day of RXP03 were decreased compared with control tumor volumes, 100 microg/day being the most effective dose. Treatment at higher dose (600 microg/day) did not significantly reduce the tumor size as compared to control. Short treatments with RXP03 100 microg/day, 3 to 7 days after C(26) inoculation, were more effective on tumor growth than continuous treatment over 18 days. Strikingly, RXP03 treatment started 6 days after the C(26) injection and continued until day 18 led to stimulation of tumor growth, as compared to control. These paradoxical effects, depending on the RXP03 treatment schedule, underline the need to define carefully the spatiotemporal function of each MMP at various stages of tumor growth to achieve optimal therapeutic effects by MMP inhibitor treatment.

Dual stromelysin-3 function during natural mouse mammary tumor virus-ras tumor progression.

In human carcinomas, stromelysin-3/matrix metalloproteinase 11 (ST3, MMP11) expression by nonmalignant fibroblastic cells located in the immediate vicinity of cancer cells is a bad prognostic factor. Using mouse models of primary tumors, it has been demonstrated that ST3 is a key player during local invasion, favoring cancer cell survival in connective tissue through an antiapoptotic function. To investigate the ST3 impact on additional phases of cancer cell invasion, we developed mammary gland cancer prone MMTV-ras transgenic mice in wild-type (ras+/+;ST3+/+) or ST3-deficient (ras+/+;ST3-/-) genotype and studied their whole natural cancer history. The tumor-free survival and delay between the first ras oncogenic hit and primary tumor appearance increased in ras+/+;ST3-/- mice (P < 0.000001 and <0.0000007, respectively). A systematic search for occult primary tumors and metastases revealed, in addition to a lower total number and size of primary tumors (P < 0.02), an unexpected higher number of metastases (P < 0.01) in ras+/+;ST3-/- mice. Moreover, for a similar number and size of primary invasive tumors, ras+/+;ST3-/- mice developed more metastases, indicating that the cancer cells evolving in ST3-deficient stroma have an increased potential to hematogenous dissemination. We conclude that the ST3 microenvironment is a consistently active partner of invading cancer cells but that its function differs throughout cancer progression, being tumor enhancer or repressor in processes leading to local or distal invasion. Such a dual effect for an MMP might shed light, at least partially, for the absence of survival benefit for patients included in anti-MMP clinical trials.

Metastatic lymph node 64 (MLN64), a gene overexpressed in breast cancers, is regulated by Sp/KLF transcription factors.

MLN64, is invariably coamplified and coexpressed with erbB-2 in breast cancers. The human MLN64 and ERBB2 genes are positioned at less than 50 kb from each other, on chromosome 17q12. To understand the molecular basis of MLN64 overexpression in cancer, the genomic region containing the MLN64 and ERBB2 genes was isolated and mapped. The two genes, DARPP32 and Telethonin, flanking MLN64 respectively on its centromeric and telomeric sides, although coamplified, are not overexpressed in breast cancer cells, indicating that gene amplification is not sufficient to allow overexpression. The MLN64 minimal promoter was isolated and found to be a housekeeping gene promoter containing four potential Sp1 binding elements. Using Sp1-deficient Drosophila SL2 cells, MLN64 promoter activity was induced in a dose-dependent manner by exogenous Sp1 addition. Furthermore, mutation of each individual Sp1 element resulted in a significant decrease in reporter gene activity, indicating that all the Sp1 binding elements are functional and act together to promote gene expression. Since the ERBB2 promoter is also positively regulated by Sp1, this study indicates that MLN64 and ERBB2 genes share common transcriptional controls together with a physical link on chromosome 17q. We speculate that, in addition to the oncogenic potential of erbB-2 overexpression, the unbalanced action of MLN64 contributes to the poor clinical outcome of breast tumors bearing this amplified region.