Tissue transglutaminase regulates focal adhesion kinase/AKT activation by modulating PTEN expression in pancreatic cancer cells.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) progresses rapidly and exhibits profound resistance to treatment. We recently reported that a great majority of PDAC tumors and tumor cell lines express elevated levels of tissue transglutaminase (TG2). Here, we provide first evidence that TG2 expression in PDAC cells results in constitutive activation of focal adhesion kinase/AKT by modulating the expression of the tumor suppressor phosphatase PTEN. EXPERIMENTAL DESIGN: Using PDAC cell lines, we determined the effect of TG2 overexpression on PTEN stability and functions. We confirmed the correlation between TG2 expression and PTEN levels in a few (n=51) PDAC tumor samples. RESULTS: We observed that expression of TG2 is inversely correlated with PTEN expression in PDAC cells. Ectopic expression of TG2 inhibited PTEN phosphorylation and promoted its degradation by ubiquitin-proteasomal pathway. Conversely, down-regulation of TG2 by small interfering RNA up-regulated PTEN expression. Clinical relevance of these results was evident in an athymic nude mouse model where down-regulation of endogenous TG2 caused a significant retardation in PDAC xenograft growth. Importantly, the analysis of 51 tumor samples from patients with stage II PDAC revealed that overexpression of TG2 was associated with loss of PTEN expression (P=0.023; odds ratio, 4.1). In multivariate analysis, TG2-mediated loss of PTEN was a prognostic factor for overall survival in patients with stage II pancreatic ductal carcinoma independent of tumor stage/lymph node status and tumor differentiation (P=0.01). CONCLUSION: TG2 expression in PDAC promotes degradation of PTEN by ubiquitin-proteasomal pathway and results in constitutive activation of focal adhesion kinase/AKT cell survival signaling.

Tissue transglutaminase inhibits autophagy in pancreatic cancer cells.

Elevated expression of tissue transglutaminase (TG2) in cancer cells has been implicated in the development of drug resistance and metastatic phenotypes. However, the role and the mechanisms that regulate TG2 expression remain elusive. Here, we provide evidence that protein kinase Cdelta (PKCdelta) regulates TG2 expression, which in turn inhibits autophagy, a type II programmed cell death, in pancreatic cancer cells that are frequently insensitive to standard chemotherapeutic agents. Rottlerin, a PKCdelta-specific inhibitor, and PKCdelta small interfering RNA (siRNA) down-regulated the expression of TG2 mRNA and protein and induced growth inhibition without inducing apoptosis in pancreatic cancer cells. Inhibition of PKCdelta by rottlerin or knockdown of TG2 protein by a TG2-specific siRNA resulted in a marked increase in autophagy shown by presence of autophagic vacuoles in the cytoplasm, formation of the acidic vesicular organelles, membrane association of microtubule-associated protein 1 light chain 3 (LC3) with autophagosomes, and a marked induction of LC3-II protein, important hallmarks of autophagy, and by electron microscopy. Furthermore, inhibition of TG2 by rottlerin or by the siRNA led to accumulation of green fluorescent protein (GFP)-LC3-II in autophagosomes in pancreatic cancer cells transfected with GFP-LC3 (GFP-ATG8) expression vector. Knockdown of Beclin-1, a specific autophagy-promoting protein and the product of Becn1 (ATG6), inhibited rottlerin-induced and TG2 siRNA-induced autophagy, indicating that Beclin-1 is required for this process. These results revealed that PKCdelta plays a critical role in the expression of TG2, which in turn regulates autophagy. In conclusion, these results suggest a novel mechanism of regulation of TG2 and TG2-mediated autophagy in pancreatic cancer cells.

Increased expression of tissue transglutaminase in pancreatic ductal adenocarcinoma and its implications in drug resistance and metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases and is virtually incurable. The molecular mechanisms that contribute to the intrinsic resistance of PDAC to various anticancer therapies are not well understood. Recently, we have observed that several drug-resistant and metastatic tumors and tumor cell lines expressed elevated levels of tissue transglutaminase (TG2). Because PDAC exhibits inherent resistance to various drugs, we determined the constitutive expression of TG2 in 75 PDAC and 12 PDAC cell lines. Our results showed that 42 of 75 (56%) PDAC tumor samples expressed higher basal levels of TG2 compared with the normal pancreatic ducts [odds ratio (OR), 2.439; P = 0.012]. The increased expression of TG2 in PDAC was strongly associated with nodal metastasis (OR, 3.400; P = 0.017) and lymphovascular invasion (OR, 3.055; P = 0.045). Increased expression of TG2 was also evident in all 12 cell lines examined. The elevated expression of TG2 in PDAC cell lines was associated with gemcitabine resistance and increased invasive potential. Overexpression of catalytically active or inactive (C(277)S mutant) TG2 induced focal adhesion kinase (FAK) activation and augmented invasive functions in the BxPC-3 cell line. Conversely, down-regulation of TG2 by small interfering RNA attenuated FAK phosphorylation. Immunoprecipitation and confocal microscopy data revealed that TG2 was associated with FAK protein in PDAC cells. The activated FAK colocalized with TG2 at focal adhesion points. These results show for the first time that elevated expression of TG2 can induce constitutive activation of FAK and thus may contribute to the development of drug resistance and invasive phenotypes in PDAC.

Implications of tissue transglutaminase expression in malignant melanoma.

Human malignant melanoma is a highly aggressive form of cancer; the 5-year survival rate in patients with stage III or IV disease is <5%. In patients with metastatic melanoma, systemic therapy becomes ineffective because of the high resistance of melanoma cells to various anticancer therapies. We have found previously that development of the drug resistance and metastatic phenotypes in breast cancer cells is associated with increased tissue transglutaminase (TG2) expression. In the study reported here, we investigated TG2 expression and its implications in metastatic melanoma. We found that metastatic melanoma cell lines expressed levels of TG2 up to 24-fold higher than levels in radial growth phase of primary melanoma cell lines. Activation of endogenous TG2 by the calcium ionophore A23187 induced a rapid and strong apoptotic response in A375 cells and A23187-induced apoptosis could be blocked by TG2-specific inhibitors. These findings indicated that activation of endogenous TG2 could serve as a strategy for inducing apoptosis in malignant melanomas. Importantly, tumor samples from patients with malignant melanomas showed strong expression of TG2, suggesting that TG2 expression is selectively up-regulated during advanced developmental stages of melanoma. We observed that 20% to 30% of TG2 protein was present on cell membranes in association with beta1 and beta5 integrins. This association of TG2 with cell surface integrins promoted strong attachment of A375 cells to fibronectin-coated surfaces, resulting in increased cell survival in serum-free medium. Inhibition of TG2 by small interfering RNA inhibited fibronectin-mediated cell attachment and cell survival functions in A375 cells. Overall, our results suggest that TG2 expression contributes to the development of chemoresistance in malignant melanoma cells by exploiting integrin-mediated cell survival signaling pathways.

Tissue transglutaminase: from biological glue to cell survival cues.

Tissue transglutaminase (TG2, EC 2.3.2.13) is a ubiquitous enzyme that catalyzes Ca2+-dependent post-translational modification of proteins by inserting highly stable (epsilon-[gamma-glutamyl] lysine) isopeptide bonds or by conjugating polyamines at selected peptide-bound glutamine residues. The TG2-catalyzed cross-linked products (generally high molecular mass scaffold of proteins) are of great physiological significance; they are highly stable and resistant to mechanical, chemical and proteolytic degradation. The accumulation of isopeptide bonds can be observed in skin, hair and during blood clotting and wound healing. In addition to transamidation activity, TG2 also exhibits GTPase activity and in response to certain agonist hormones can serve as a signal transducing G protein. Although predominantly a cytosolic protein, TG2 can translocate to the nucleus with the help of importin alpha-3 protein or to the membranes in association with integrins. Moreover, TG2 can also be secreted outside the cell (by yet unknown mechanism) where it crosslinks proteins of the extracellular matrix (ECM) and promotes cell adhesion and spreading. Another important property of TG2 is that it has high binding-affinity for the ECM component protein, fibronectin and thus can promote interaction between cell surface integrin with fibronectin. In this review, we discuss the implications of increased TG2 expression in drug-resistant and metastatic cancer cells and that how TG2 expression can contribute in the development of these phenotypes.

Prognostic significance of tissue transglutaminase in drug resistant and metastatic breast cancer.

PURPOSE: Drug resistance and metastasis pose major impediments in the successful treatment of cancer. We previously reported that multidrug-resistant breast cancer cells exhibit high levels of tissue transglutaminase (TG2; EC 2.3.2.13). Because the drug-resistant and metastatic phenotypes are thought to share some common pathways, we sought to determine whether metastatic breast cancer cells express high levels of TG2. EXPERIMENTAL DESIGN: The metastatic breast cancer cell line MDA-MB-231 and the sublines derived from it were tested for TG2 expression. Similarly, several sublines derived from an immortal but normal breast epithelial cell line, MCF10A, representing various stages in breast cancer progression were studied for TG2 expression. The primary and nodal tumor samples from 30 patients with breast cancer were also studied for TG2 expression. RESULTS: The MDA-MB-231 cells expressed high basal levels of TG2. Two clones derived from this cell line, MDA231/cl.9 and MDA231/cl.16, showed a 10- to 15-fold difference in TG2 level. TG2-deficient MDA231/cl.9 cells exhibited higher sensitivity to doxorubicin and were less invasive than were the TG2-sufficient MDA231/cl.16 cells. The MCF10A-derived sublines had increased TG2 expression as they advanced from noninvasive to an invasive phenotype. Importantly, the metastatic lymph node tumors from patients with breast cancer showed significant higher levels of TG2 expression compared with the primary tumors from the same patients. CONCLUSIONS: TG2 expression is up-regulated in drug-resistant and metastatic breast cancer cells, and it can serve as a valuable prognostic marker for these phenotypes.

Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor.

Many studies have supported a role for inflammation in prostate tumour growth. However, the contribution of inflammation to the development of castration-resistant prostate cancer remains largely unknown. Based on observations that aberrant expression of the proinflammatory protein tissue transglutaminase (TG2) is associated with development of drug resistance and metastatic phenotype in multiple cancer types, we determined TG2 expression in prostate cancer cells. Herein we report that human prostate cancer cell lines with low expression of androgen receptor (AR) had high basal levels of TG2 expression. Also, overexpression of TG2 negatively regulated AR mRNA and protein expression and attenuated androgen sensitivity of prostate cancer cells. TG2 expression in prostate cancer cells was associated with increased invasion and resistance to chemotherapy. Mechanistically, TG2 activated nuclear factor (NF)-κB and induced epithelial-mesenchymal transition. TG2/NF-κB-mediated decrease in AR expression resulted from transcriptional repression involving cis-interaction of NF-κB in a complex with TG2 with the 5'-untranslated region of AR. Negative regulation of AR could be partially abrogated by repression of TG2 or NF-κB (p65/RelA) by gene-specific small interfering RNA. These results suggested that a novel pathway links androgen dependence with TG2-regulated inflammatory signalling and hence may make TG2 a novel therapeutic target for the prevention and treatment of castration-resistant prostate cancer.

Combining erlotinib and cetuximab is associated with activity in patients with non-small cell lung cancer (including squamous cell carcinomas) and wild-type EGFR or resistant mutations.

Preclinical data suggest that combined EGF receptor (EGFR) targeting with an EGFR tyrosine kinase inhibitor and an anti-EGFR monoclonal antibody may be superior over single-agent targeting. Therefore, as part of a phase I study, we analyzed the outcome of 20 patients with non-small cell lung cancer treated with the combination of erlotinib and cetuximab. EGFR mutation status was ascertained in a Clinical Laboratory Improvement Amendment-approved laboratory. There were 10 men; median number of prior therapies was five. Overall, two of 20 patients (10%) achieved partial response (PR), one of whom had a TKI-resistant EGFR insertion in exon 20, time to treatment failure (TTF) = 24+ months, and the other patient had squamous cell histology (EGFR wild-type), TTF = 7.4 months. In addition, three of 20 patients (15%) achieved stable disease (SD) ≥6 six months (one of whom had wild-type EGFR and squamous cell histology, and two patients had an EGFR TKI-sensitive mutation, one of whom had failed prior erlotinib therapy). Combination therapy with ertotinib plus cetuximab was well tolerated. The most common toxicities were rash, diarrhea, and hypomagnesemia. The recommended phase II dose was erlotinib 150 mg oral daily and cetuximab 250 mg/m(2) i.v. weekly. In summary, erlotinib and cetuximab treatment was associated with SD ≥ six months/PR in five of 20 patients with non-small cell lung cancer (25%), including individuals with squamous histology, TKI-resistant EGFR mutations, and wild-type EGFR, and those who had progressed on prior erlotinib after an initial response. This combination warrants further study in select populations of non-small cell lung cancer.

Revisiting clinical trials using EGFR inhibitor-based regimens in patients with advanced non-small cell lung cancer: a retrospective analysis of an MD Anderson Cancer Center phase I population.

PURPOSE: Single-agent EGFR inhibitor therapy is effective mainly in patients with lung cancer and EGFR mutations. Treating patients who develop resistance, or who are insensitive from the outset, often because of resistant mutations, other aberrations or the lack of an EGFR mutation, probably requires rational combinations. We therefore investigated the outcome of EGFR inhibitor-based combination regimens in patients with heavily-pretreated non-small cell lung cancer (NSCLC) referred to a Phase I Clinic. METHODS: We reviewed the electronic records of patients with NSCLC treated with an EGFR inhibitor-based combination regimen: erlotinib and cetuximab; erlotinib, cetuximab and bevacizumab; erlotinib and dasatinib; erlotinib and bortezomib; or cetuximab and sirolimus. RESULTS: EGFR mutations were detected in 16% of patients (21/131). EGFR inhibitor-based combination regimens were administered to 15 patients with EGFR-mutant NSCLC and 24 with EGFR wild-type disease. Stable disease (SD) ≥6 months/partial remission (PR) was attained in 20% of EGFR-mutant patients (3/15; two with sensitive mutations and secondary resistance to prior erlotinib, and one with a resistant mutation), as well as 26% of evaluable patients (5/19) with wild-type disease. One of three evaluable patients with squamous cell histology achieved SD for 26.5 months (EGFR wild-type, TP53-mutant, regimen=erlotinib, cetuximab and bevacizumab). CONCLUSIONS: Eight of 34 evaluable patients (24%) with advanced, refractory NSCLC evaluable for response achieved SD ≥6 months/PR (PR=3; SD ≥6 months=5) on EGFR inhibitor-based combination regimens (erlotinib, cetuximab; erlotinib, cetuximab and bevacizumab; and, erlotinib, bortezomib), including patients with secondary resistance to single-agent EGFR inhibitors, resistant mutations, wild-type disease, and, squamous histology.

Clinical and biological significance of tissue transglutaminase in ovarian carcinoma.

Tissue type transglutaminase (TG2) is a unique multifunctional protein that plays a role in many steps in the cancer metastatic cascade. Here, we examined the clinical (n = 93 epithelial ovarian cancers) and biological (in vitro adhesion, invasion, and survival and in vivo therapeutic targeting) significance of TG2 in ovarian cancer. The overexpression of TG2 was associated with significantly worse overall patient survival in both univariate and multivariate analyses. Transfection of TG2 into SKOV3ip1 cells promoted attachment and spreading on fibronectin-coated surfaces and increased the in vitro invasive potential of these cells. Conversely, TG2 silencing with small interfering RNA (siRNA) of HeyA8 cells significantly decreased the invasive potential of the cells and also increased docetaxel-induced cell death. In vivo therapy experiments using chemotherapy-sensitive (HeyA8) and chemotherapy-resistant (HeyA8-MDR and RMG2) models showed significant antitumor activity both with TG2 siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine alone and in combination with docetaxel chemotherapy. This antitumor activity was related to decreased proliferation and angiogenesis and increased tumor cell apoptosis in vivo. Taken together, these findings indicate that TG2 overexpression is an adverse prognostic factor in ovarian carcinoma and TG2 targeting may be an attractive therapeutic approach.

Tissue transglutaminase induces the release of apoptosis inducing factor and results in apoptotic death of pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignant disease with poor long-term survival rates. Major reason for poor disease outcome is the profound intrinsic resistance of PDAC cells to currently available treatment regimens. We recently found that a great majority of PDAC tumors and tumor cell lines express high basal level of tissue transglutaminase (TG2), a multifunctional protein implicated in apoptosis, cell attachment, cell survival, and cell motility functions. Based on these observations, we hypothesized that activation of endogenous TG2 can induce spontaneous apoptosis in PDAC cells. The results obtained suggested that activation of endogenous TG2 by calcium ionophore A23187 induced rapid and spontaneous apoptosis in PDAC cells. TG2-induced apoptosis was associated with release of apoptosis-inducing factor (AIF). The release of AIF from mitochondria led to its translocation to the nucleus and subsequent apoptosis of PDAC cells in caspase-independent manner. In conclusion, our results provide first evidence that TG2 can induce apoptosis in PDAC cells in an AIF-dependent and caspase-independent manner.

Overexpression of tissue transglutaminase leads to constitutive activation of nuclear factor-kappaB in cancer cells: delineation of a novel pathway.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-kappaB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Based on our previous results showing that drug-resistant and metastatic cancer cells have high levels of tissue transglutaminase (TG2) expression and that this expression can confer chemoresistance to certain types of cancer cells, we hypothesized that TG2 contributes to constitutive activation of NF-kappaB. Numerous lines of evidence showed that overexpression of TG2 is linked with constitutive activation of NF-kappaB. Tumor cells with overexpression of TG2 exhibited increased levels of constitutively active NF-kappaB. Activation of TG2 led to activation of NF-kappaB; conversely, inhibition of TG2 activity inhibited activation of NF-kappaB. Similarly, ectopic expression of TG2 caused activation of NF-kappaB, and inhibition of expression of TG2 by small interfering RNA abolished the activation of NF-kappaB. Our results further indicated that constitutive NF-kappaB reporter activity in pancreatic cancer cells is not affected by dominant-negative I kappaB alpha. Additionally, coimmunoprecipitation and confocal microscopy showed that I kappaB alpha is physically associated with TG2. Lastly, immunohistochemical analysis of pancreatic ductal carcinoma samples obtained from 61 patients further supported a strong correlation between TG2 expression and NF-kappaB activation/overexpression (P = 0.0098, Fisher's exact test). We conclude that TG2 induces constitutive activation of NF-kappaB in tumor cells via a novel pathway that is most likely independent of I kappaB alpha kinase. Therefore, TG2 may be an attractive alternate target for inhibiting constitutive NF-kappaB activation and rendering cancer cells sensitive to anticancer therapies.

Antitumor metallothiosemicarbazonates: structure and antitumor activity of palladium complex of phenanthrenequinone thiosemicarbazone.

The crystal structure of the potential antitumor metal compound, viz. chloro, mono(phenanthrenequinone thiosemicarbazonato) palladium(II) dimethyl formamide solvate, is reported. The central palladium(II) atom is in a square planar environment provided by the tridentate, monoanionic thiosemicarbazone ligand and the ancillary chloride ion. The compound exhibited remarkable activity against drug-sensitive and drug-resistant breast cancer cell lines and was relatively nontoxic toward the normal mammary epithelial cells. The drug-induced killing effect against breast cancer cell lines was predominantly mediated via apoptosis, a physiologic form of cell death.