Role of EGFR family receptors in proliferation of squamous carcinoma cells induced by wound healing fluids of head and neck cancer patients.

BACKGROUND: Mounting evidence suggests that recurrence of resected head and neck squamous cell carcinomas (HNSCCs) is due to the outgrowth of unrecognized residual tumor cells as well as to the premalignant and/or precursor-field epithelial cells. We studied the impact of processes triggered by HNSCC surgery in stimulating both residual tumor cells [demonstrated to overexpress epidermal growth factor receptor (EGFR)], and premalignant cells surrounding the resected lesion. PATIENTS AND METHODS: EGFR expression/activation by immunohistochemistry/biochemistry and gene status by FISH were investigated in 23 primary HNSCCs and surrounding tissues. The ability to induce cell proliferation of wound healing drainages collected from 12 relapsed and 11 not relapsed patients was evaluated by a colorimetric assay in squamous cell carcinoma cell lines A431 (carrying EGFR amplification) and CAL27 (carrying three EGFR copies) in the presence/absence of EGFR therapeutic inhibitors. RESULTS: Primary tumors showed intermediate/high EGFR expression (91%), EGFR phosphorylation and EGFR-positive FISH (35%). Normal, metaplastic and dysplastic epithelium surrounding the resected tumor displayed EGFR overexpression. EGFR activation and gene amplification were observed in normal and dysplastic epithelium, respectively. Each tested wound healing drainage induced the cells to proliferate and the proliferation was significantly higher in relapsed compared with not relapsed HNSCC patients (P = 0.02 and P = 0.03). Anti-EGFR treatments inhibited the drainage-induced proliferation, with the highest inhibitory efficiency by cetuximab on A431 cells, while CAL27 cell growth was more efficiently inhibited by tyrosine kinase inhibitors. CONCLUSIONS: Surgery could favor the proliferation of cells showing EGFR overexpression/activation/amplification such as residual tumor cells and/or precursor-field epithelial cells already present after surgery. Treatment with anti-EGFR reagents inhibits wound-induced stimulation, according to the EGFR family status.

Cetuximab in recurrent and/or metastatic salivary gland carcinomas: A phase II study.

EGFR overexpression in salivary gland carcinomas provides the rational for the investigation of anti-EGFR treatments in recurrent and/or metastatic salivary gland cancers (RMSGCs). The activity of cetuximab in terms of clinical benefit rate (CBR) defined as the occurrence of objective response (CR or PR) or stable disease (SD) for >or=6months was investigated. From April to December 2005, 30 patients [23 adenoid cystic carcinoma (ACC) and 7 non-ACC] were treated with cetuximab at 400mg/m(2)/week followed by 250mg/m(2)/week until progression, major toxicity or voluntary discontinuation. EGFR expression and gene status were retrospectively analyzed by immunocytochemistry and fluorescence in situ hybridization, respectively. A median of 14 courses of cetuximab (range 5-54) were infused. Skin toxicity was the main adverse event. Cetuximab provides a CBR in 50% (95% CL, 31 to 69%) of cases. None tumor sample showed EGFR gene amplification and an increased EGFR copy number was observed in 12% of samples, all ACC. Skin rash >or=G2, EGFR overexpression and EGFR copy number were not statistically correlated to CB. In RMSGCs further evaluations of EGFR targeting agents are advisable and should take place by appropriate tumor biological selection, differentiating ACC from non-ACC.

PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients.

BACKGROUND: It has been reported that KRAS mutations (and to a lesser extent KRAS mutations with the BRAF V600E mutation) negatively affect response to anti-epidermal growth factor receptor (EGFR) mAbs in metastatic colorectal cancer (mCRC) patients, while the biological impact of the EGFR pathway represented by PI3K/PTEN/AKT on anti-EGFR treatment is still not clear. PATIENTS AND METHODS: We analysed formalin-fixed samples from a cohort of 32 mCRC patients treated with cetuximab by means of EGFR immunohistochemistry, EGFR and PTEN FISH analysis, and KRAS, BRAF, PI3KCA, and PTEN genomic sequencing. RESULTS: Ten (31%) of 32 patients showed a partial response to cetuximab and 22 (69%) did not [nonresponder (NR)]. EGFR immunophenotype and FISH-based gene status did not predict an anti-EGFR mAb response, whereas KRAS mutations (24%) and PI3K pathway activation, by means of PI3KCA mutations (13%) or PTEN mutation (10%)/loss (13%), were significantly restricted to, respectively, 41% and 37% of NRs. CONCLUSION: These findings suggested that KRAS mutations and PI3KCA/PTEN deregulation significantly correlate with resistance to cetuximab. In line with this, patients carrying KRAS mutations or with activated PI3K profiles can benefit from targeted treatments only by switching off molecules belonging to the downstream signalling of activated EGFR, such as mammalian target of rapamycin.

Analysis of potential receptor tyrosine kinase targets in intimal and mural sarcomas.

Primary sarcomas of the great vessels are very rare neoplasms and only a few cases have been reported. They are divided into the two broad categories of intimal or luminal and mural sarcomas. We analysed eight advanced high-grade sarcomas originating from major vessels (seven intimal and one mural sarcoma) by means of immunohistochemistry and FISH analysis for PDGFRA, PDGFRB, EGFR and KIT receptor tyrosine kinases (RTKs), together with immunoprecipitation/western blotting, sequencing of the corresponding genes, and the search for cognate ligands. The intimal sarcomas showed a wide spectrum of morphologies and immunophenotypes, whereas the mural sarcoma had common leiomyosarcomatous features. Regardless of their category, all of the cases had a PDGFRA-deregulated cytogenetic profile mainly consisting of an amplification cluster; five were also polysomic for PDGFRB, whereas three showed disomy. Six cases had a deregulated EGFR gene, and c-Kit gene status was similar to that of PDGFRA. In one case, biochemical analysis revealed the presence of activated and highly expressed PDGFRA, PDGFRB and EGFR, whereas KIT was expressed at reference level. Sequencing of the corresponding genes revealed no activating mutations in any of the analysed receptors. The cognate ligands were detected in all cases. In predictive terms, the evidence of gene amplification/high polysomy of several RTKs, together with PDGFRA, PDGFRB and EGFR expression and phosphorylation, suggests that these tumours may be sensitive to RTK-inhibiting treatments.

Involvement of H(+)-ATPase and carbonic anhydrase in inorganic carbon uptake for endosymbiont photosynthesis.

Symbiotic cnidarians absorb inorganic carbon from seawater to supply intracellular dinoflagellates with CO(2) for their photosynthesis. To determine the mechanism of inorganic carbon transport by animal cells, we used plasma membrane vesicles prepared from ectodermal cells isolated from tentacles of the sea anemone, Anemonia viridis. H(14)CO(-)(3) uptake in the presence of an outward NaCl gradient or inward H(+) gradient, showed no evidence for a Cl(-)- or H(+)- driven HCO(-)(3) transport. H(14)CO(-)(3) and (36)Cl(-) uptakes were stimulated by a positive inside-membrane diffusion potential, suggesting the presence of HCO(-)(3) and Cl(-) conductances. A carbonic anhydrase (CA) activity was measured on plasma membrane (4%) and in the cytoplasm of the ectodermal cells (96%) and was sensitive to acetazolamide (IC(50) = 20 nM) and ethoxyzolamide (IC(50) = 2.5 nM). A strong DIDS-sensitive H(+)-ATPase activity was observed (IC(50) = 14 microM). This activity was also highly sensitive to vanadate and allyl isothiocyanate, two inhibitors of P-type H(+)-ATPases. Present data suggest that HCO(-)(3) absorption by ectodermal cells is carried out by H(+) secretion by H(+)-ATPase, resulting in the formation of carbonic acid in the surrounding seawater, which is quickly dehydrated into CO(2) by a membrane-bound CA. CO(2) then diffuses passively into the cell where it is hydrated in HCO(-)(3) by a cytosolic CA.

A monocarboxylate transporter MCT1 is located at the basolateral pole of rat jejunum.

We have functionally expressed and identified a monocarboxylate transporter (MCT1) from rat jejunal enterocyte and we provide evidence for its basolateral localization. Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of a proton-lactate symporter was investigated by means of L-[14C]lactate uptake. The existence of an endogenous capacity for L-lactate transport was demonstrated; when, however, oocytes were injected with jejunal mRNA, an expressed L-lactate uptake was seen which differed from the endogenous transporter since it was significantly pH dependent. After sucrose density gradient fractionation, the highest expression of the pH-dependent lactate uptake was detected with the mRNA size fraction of about 2-3 kb in length. The substrate specificity, stereoselectivity and sensitivity to pCMBS (an organomercurial thiol reagent that modifies cysteine residues) of the expressed transport were in good agreement with results previously obtained using isolated jejunal basolateral membranes. Using the reverse transcriptase-polymerase chain reaction, the presence of mRNA coding for the MCT1 isoform was demonstrated in jejunal enterocytes. These data, together with previous results, suggest that MCT1 is a major route for lactate efflux across the basolateral membrane of rat jejunum; this is in contrast to current opinion which restricts the presence of MCT1 to the apical membrane of the whole small intestine.

Facilitated transport of lactate by rat jejunal enterocyte.

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K(m) of 39.2 +/- 4.8 mM and a Jmax of 8.9 +/- 0.7 nmoles mg protein-1 sec-1. A very small conductive pathway for L-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H(+)-lactate cotransporter, whereas in the apical membrane both H(+)-lactate and Na(+)-lactate cotransporters are present, even if they exhibit a low transport rate.

Aging and ATPase activities in rat jejunum.

In addition to the well-known (Na,K)-ATPase activity, an ouabain-insensitive Na-ATPase has been evidenced in the basolateral membrane of intestinal and renal cells from different mammals. Basolateral membranes of jejunal enterocytes from rats of different ages, i.e., very young, young, adult and old were separated by self-orienting, Percoll-gradient centrifugation. The total protein content and both Na- and (Na,K)-ATPase activities in initial homogenate and final pellets were analyzed. The dry weight of homogenate and pellet was also determined. The two ATPase activities and the protein content of the basolateral membrane fraction decrease with age when referred to the dry weight of the pellet. This diminution is also evident in the initial homogenate. The activation curve of Na-ATPase, hyperbolic in shape, gives Km and Vmax values unaffected by aging. The same behaviour is true for the kinetic parameters of (Na,K)-ATPase, which has a sigmoidal velocity curve. From these results, it seems that both Na- and (Na,K)-ATPase have the same characteristics in the basolateral membrane of the enterocyte throughout the life span of the animal, but they decrease quantitatively with aging.

Ouabain-insensitive active sodium transport in rat jejunum: evidence from ATPase activities, Na uptake by basolateral membrane vesicles and in vitro transintestinal transport.

The basolateral membrane of the jejunal enterocyte of the rat was separated by self-orienting Percoll-gradient centrifugation and further purified from brush border contamination. Pellets were analysed for Mg-, Na- and (Na,K)-ATPase activities. The uptake of 0.02 M NaCl was also followed by the rapid micro-filtration technique. Transintestinal transport of fluid and electrolytes, and cell water, Na and K were determined in the in vitro everted and incubated jejunum. There is ouabain-insensitive Na-ATPase in addition to the well-known (Na,K)-ATPase in the basolateral membrane. These are differently inhibited by furosemide and ethacrynate. Na uptake by osmotically active basolateral membrane vesicles is enhanced by ATP and a further enhancement is obtained if there is intravesicular K. The ATP effect is inhibited differently by strophanthidin, furosemide and ethacrynate. In the everted sac preparation, transintestinal transport of Na and fluid still occurs when the Na/K pump is totally inhibited by ouabain. These experimental results suggest that there is also a ouabain-insensitive Na pump, different from the Na/K pump, in the basolateral membrane.

Mechanisms of Na transport at the basolateral pole of rat enterocyte.

Basolateral membranes isolated from rat jejunal enterocytes and enriched 14 times were found present as unsealed and sealed right side out (RSO) or inside out (IO) vesicles in the ratio 2:2:1. Entrance of 1 mM Na (JNa) into basolateral membrane vesicles was measured in the presence and in the absence of 5 mM ATP by a rapid filtration technique, under different experimental conditions. JNa across the basolateral membrane is cis-inhibited by K, transstimulated by K and further stimulated by ATP (activation of the Na pump). Both K- and ATP-positive effects are also obtained by other cations: the relative stimulation sequence is K greater than Rb = NH4 greater than Cs. The ATP effect can be suppressed by vanadate and strophanthidin; moreover the K effect on JNa is not influenced by blocking the mechanism of the Na pump with ouabain, strophanthidin and adenosine 5'-[beta,gamma-imido]triphosphate. The transmembrane potential does not influence the K-stimulated JNa. In addition to the Na pump this study demonstrates the existence of an exchange mechanism between Na and K, which seems to be different from the Na pump. There appears to be no Na-K cotransport.

Ouabain-insensitive Na-ATPase activity in the basolateral membrane from rat jejunum.

1. In the basolateral membrane preparation of the rat enterocyte (jejunal tract) there is not only the well-known (Na,K)-ATPase activity, but also a ouabain-insensitive Na-ATPase. 2. The Na-ATPase is not activated by anions or other monovalent cations. As a substrate, ATP cannot be replaced by other nucleotides. 3. The Na-ATPase is insensitive to ouabain and bumetanide, inhibited partially by furosemide and totally by ethacrynate. 4. The activation of Na-ATPase at different Na concentrations shows an hyperbolic curve (Km = 15.7 +/- 2.3 mM and Vmax = 204 +/- 19 nmoles Pi/mg protein per min) different from the sigmoidal curve (Km = 9.8 +/- 1.2 mM and Vmax = 640 +/- 15 nmoles Pi/mg protein per min) shown by (Na,K)-ATPase. 5. These results are compared with the corresponding ones found in other animals and tissues in which the Na-ATPase was found. 6. The Na-ATPase activity can be interpreted as the enzymatic correspondent of a ouabain-insensitive Na pump, present in the basolateral membrane of the enterocyte different in behaviour with respect to the known Na pump.

Sodium transport in basolateral membrane vesicles from rat enterocytes.

Basolateral membranes purified from rat jejunal enterocytes and enriched 14 times in (Na, K)-ATPase, are present as unsealed and right side out (RSO) or inside out (IO) vesicles in the ratio 2:2:1, as determined by detergent activation of ATPase activity. Entrance of 1 mM Na into basolateral membrane vesicles was measured in the presence and in the absence of 5 mM ATP by a rapid filtration technique, under different experimental conditions. Carrier-mediated Na transport across the basolateral membrane can be trans-stimulated and cis-inhibited by K and further stimulated by ATP (activation of the Na pump). The ATP effect can be suppressed by vanadate and strophanthidin and enhanced by bleomycin (19% increase), which positively also acts on (Na, K)-ATPase activity (16% increase). In addition to the Na pump this study demonstrates the existence of a carrier-mediated Na transport trans-stimulated by K. There appears to be no cotransport of Na-K.

The basolateral membrane of rat enterocyte: its purification from brush border contamination.

Basolateral membranes obtained by self-orienting Percoll-gradient centrifugation were treated with 5 mM CaCl2 to minimize the cross-contamination by brush border membranes. From marker enzyme-specific activities it was calculated that in this preparation the basolateral/brush border membrane ratio was 22.6. A low L-glucose permeability across basolateral membrane vesicles together with ATP-dependent sodium uptake was observed.

Effects of the stilbene derivatives SITS and DIDS on intestinal ATPase activities.

4-Acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS) and 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) effects have been tested both in the basolateral membranes (BLMs) of jejunum enterocytes and in the same intestinal tract, everted and incubated "in vitro". Total and (Na,K)-ATPase activities of BLMs are inhibited in a similar way by the two disulfonic stilbenes as well as the fluid transintestinal transport in the everted intestine; on the contrary cell Na and K are unaffected. SITS and DIDS inhibition of (Na,K)-ATPase seems to take place at the cytoplasmic side of the BLM.