Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway.

Epidermal growth factor receptor (EGFR) exists in the nucleus of highly proliferative cells where it functions as a transcription factor. Although EGFR has transactivational activity, it lacks a DNA binding domain and, therefore, may require a DNA binding transcription cofactor for its transcriptional function. Here, we report that EGFR physically interacts with signal transducers and activators of transcription 3 (STAT3) in the nucleus, leading to transcriptional activation of inducible nitric oxide synthase (iNOS). In breast carcinomas, nuclear EGFR positively correlates with iNOS. This study describes a mode of transcriptional control involving cooperated efforts of STAT3 and nuclear EGFR. Our work suggests that the deregulated iNOS/NO pathway may partly contribute to the malignant biology of tumor cells with high levels of nuclear EGFR and STAT3.

Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2.

Pathological expression of human ErbB-2 protein, also known as HER-2, is common in many types of cancer. ErbB-2 is a member of the EGF receptor tyrosine kinase family and has been rigorously studied as a signaling molecule on the cell membrane. Here, we report that ErbB-2 is also expressed in the nucleus in cultured cells as well as primary tumor tissues. Nuclear ErbB-2 was found to associate with multiple genomic targets in vivo, including the cyclooxygenase enzyme COX-2 gene promoter. ErbB-2 forms a complex at a specific nucleotide sequence of the COX-2 promoter and is able to stimulate its transcription. This study demonstrates the presence of ErbB-2 in the nucleus and identifies the function of ErbB-2 as a transcriptional regulator.

MDM2 promotes cell motility and invasiveness by regulating E-cadherin degradation.

Gene amplification and protein overexpression of MDM2, which is often found in certain types of cancers, indicate that MDM2 plays an important role in tumorigenesis. Interestingly, several clinical reports have demonstrated that amplification of the MDM2 gene correlates with the metastatic stage. Using an antibody array assay, we identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin is a substrate for the MDM2 E3 ubiquitin ligase. We demonstrate that MDM2 interacts in vivo with E-cadherin, resulting in its ubiquitination and degradation. This regulation appears to be clinically relevant, as we found a significant correlation between high MDM2 and low E-cadherin protein levels in resected tumor specimens recovered from breast cancer patients with lymph node metastases. Ectopic expression of MDM2 in breast cancer cells was found to disrupt cell-cell contacts and enhance cell motility and invasive potential. We found that E-cadherin and MDM2 colocalized on the plasma membrane and in the early endosome, where ubiquitin moieties were attached to E-cadherin. Blocking endocytosis with dominant-negative mutants of dynamin abolished the association of MDM2 with E-cadherin, prevented E-cadherin degradation, and attenuated cell motility as observed by fluorescence microscopy. Thus, we provide evidence to support a novel role for MDM2 in regulating cell adhesions by a mechanism that involves degrading and down-regulating the expression of E-cadherin via an endosome pathway. This novel MDM2-regulated pathway is likely to play a biologically relevant role in cancer metastasis.

Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor.

The cell membrane receptor ErbB-2 migrates to the nucleus. However, the mechanism of its nuclear translocation is unclear. Here, we report a novel mechanism of its nuclear localization that involves interaction with the transport receptor importin beta1, nuclear pore protein Nup358, and a host of players in endocytic internalization. Knocking down importin beta1 using small interfering RNA oligonucleotides or inactivation of small GTPase Ran by RanQ69L, a dominant-negative mutant of Ran, causes a nuclear transport defect of ErbB-2. Mutation of a putative nuclear localization signal in ErbB-2 destroys its interaction with importin beta1 and arrests nuclear translocation, while inactivation of nuclear export receptor piles up ErbB-2 within the nucleus. Additionally, blocking of internalization by a dominant-negative mutant of dynamin halts its nuclear localization. Thus, the cell membrane-embedded ErbB-2, through endocytosis using the endocytic vesicle as a vehicle, importin beta1 as a driver and Nup358 as a traffic light, migrates from the cell surface to the nucleus. This novel mechanism explains how a receptor tyrosine kinase on the cell surface can be translocated into the nucleus. This pathway may serve as a general mechanism to allow direct communication between cell surface receptors and the nucleus, and our findings thus open a new era in understanding direct trafficking between the cell membrane and nucleus.

Sex-determining region Y box 4 is a transforming oncogene in human prostate cancer cells.

Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene.

Apoptosis signalling mechanisms in human cancer cells induced by Calphostin-PDT.

Photodynamic therapy (PDT) is a promising treatment that is approved by the US FDA for the treatment of oesophageal and lung cancer as well as for age-related macular degeneration. In this study, using standard tissue culture techniques, the photo cytotoxicity and apoptotic mechanisms of Calphostin C (Cal C), a perylenequinone microbial compound in combination with visible light dose was examined in different tumor cell lines. Our results demonstrated both a time and drug-light dose dependence in Cal-C-PDT induced photo toxicity and apoptotic cell death. The induction of apoptosis by Cal C-PDT was found to transit to necrotic cell death at higher drug and light doses. The detection of apoptosis in irradiated tumor cells was performed using various approaches including cell morphology analysis, flow cytometry [DNA fragmentation and phosphatidylserine (PS) externalization] and biochemical assays (activation of caspases). Time-course analysis of Cal C cellular uptake and distribution showed a rapid increase within the cellular compartments. The activation of caspases and nuclear fragmentation was evidenced at a maximum time point of 3 h after irradiation. By the use of specific caspase substrates, significant activation of caspase-8 and -3 was found. Mitochondrial involvement during Cal C-PDT-induced apoptosis was proven by a rapid reduction of the mitochondrial membrane potential. Furthermore, Cal C-PDT also enhanced FasL expression, which then induced Fas signalling-dependent cell death in NPC and colon cancer cell lines tested. Our results contribute to a deeper understanding of the processes involved in apoptotic cell death following photodynamic treatment with Cal C.

Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer.

Epidermal growth factor receptor (EGFR) has been detected in the nucleus of cancer cells and primary tumors for decades. While localized in the nucleus, EGFR functions as a transcriptional regulator resulting in the activation of the cyclin D1 gene. Despite nuclear accumulation of EGFR is linked to increased DNA synthesis and proliferative potential, the pathological significance of nuclear EGFR, however, remains uninvestigated. Furthermore, expression of EGFR has not provided a consistent predictive value for survival of breast cancer patients. Here, we analyzed 130 breast carcinomas via immunohistochemical analyses for the levels of nuclear and non-nuclear EGFR. We found 37.7% of the cohort immunostained positively for nuclear EGFR and 6.9% with high levels of expression. Importantly, Kaplan-Meier survival analysis and log-rank test revealed a significant inverse correlation between high nuclear EGFR and overall survival (P = 0.009). Expression of nuclear EGFR correlated positively with increased levels of cyclin D1 and Ki-67, both are indicators for cell proliferation. In contrast, expression of non-nuclear EGFR did not significantly correlate with those of cyclin D1 and Ki-67 or the overall survival rate. In addition, we analyzed 37 oral squamous carcinomas for EGFR expression and found 24.3% of the cases to contain moderate/high levels of nuclear EGFR. Taken together, our findings indicate pathological significance of nuclear EGFR and may have important clinical implication.

Cross-platform expression profiling demonstrates that SV40 small tumor antigen activates Notch, Hedgehog, and Wnt signaling in human cells.

BACKGROUND: We previously analyzed human embryonic kidney (HEK) cell lines for the effects that simian virus 40 (SV40) small tumor antigen (ST) has on gene expression using Affymetrix U133 GeneChips. To cross-validate and extend our initial findings, we sought to compare the expression profiles of these cell lines using an alternative microarray platform. METHODS: We have analyzed matched cell lines with and without expression of SV40 ST using an Applied Biosystems (AB) microarray platform that uses single 60-mer oligonucleotides and single-color quantitative chemiluminescence for detection. RESULTS: While we were able to previously identify only 456 genes affected by ST with the Affymetrix platform, we identified 1927 individual genes with the AB platform. Additional technical replicates increased the number of identified genes to 3478 genes and confirmed the changes in 278 (61%) of our original set of 456 genes. Among the 3200 genes newly identified as affected by SV40 ST, we confirmed 20 by QRTPCR including several components of the Wnt, Notch, and Hedgehog signaling pathways, consistent with SV40 ST activation of these developmental pathways. While inhibitors of Notch activation had no effect on cell survival, cyclopamine had a potent killing effect on cells expressing SV40 ST. CONCLUSIONS: These data show that SV40 ST expression alters cell survival pathways to sensitize cells to the killing effect of Hedgehog pathway inhibitors.

Betulinic Acid: Recent Advances in Chemical Modifications, Effective Delivery, and Molecular Mechanisms of a Promising Anticancer Therapy.

An important method of drug discovery is examination of diverse life forms, including medicinal plants and natural products or bioactive compounds isolated from these sources. In cancer research, lead structures of compounds from natural sources can be used to design novel chemotherapies with enhanced biological properties. Betulinic acid (3ß-hydroxy-lup-20(29)-en-28-oic acid or BetA) is a naturally occurring pentacyclic triterpene with a wide variety of biological activities, including potent antitumor properties. Non-malignant cells and normal tissues are not affected by BetA. Because BetA exerts its effects directly on the mitochondrion and triggers death of cancerous cells, it is an important alternative when certain chemotherapy drugs fail. Mitochondrion-targeted agents such as BetA hold great promise to circumvent drug resistance in human cancers. BetA is being developed by a large network of clinical trial groups with the support of the U.S. National Cancer Institute. This article discusses recent advances in research into anticancer activity of BetA, relevant modes of delivery, and the agent's therapeutic efficacy, mechanism of action, and future perspective as a pipeline anticancer drug. BetA is a potentially important agent in cancer therapeutics.

Mechanisms of action of phenanthroperylenequinones in photodynamic therapy (review).

Despite the age-old belief that most anti-cancer agents kill tumor cells by necrosis, recent findings have demonstrated that photosensitizers could also kill tumor cells by triggering genetically programmed series of events termed apoptosis. Cell death by apoptosis is a very neat way to eliminate unwanted cells: no traces are left and the cell contents are never released or accessible to the immune system. Hence there is no inflammation. This is in contrast to death by necrosis. Under these conditions, normally the cell swells and then, when membrane integrity comes under attack, the cell collapses like a balloon and the contents spill out into the extracellular milieu. This may result in an inflammatory response. Because of the relatively clean nature of the apoptotic process, it is desirable to identify compounds that effectively activate the apoptotic pathway. Photodynamic therapy (PDT), a new mode of treatment, is based on the combined use of light-absorbing compounds and light irradiation. Recent developments in understanding the mechanisms of the PDT effect of photosensitizers indicate that a critical factor in the success of the agent is the ability to induce apoptosis in the malignant cell population. Hypericin and Hypocrellins are perylquinones, which are novel natural photosensitizers characterized by high absorption around 470 nm and high singlet oxygen yield. To study the signaling mechanism in vitro we have investigated uptake kinetics, intracellular localization, mode of cell death and mechanisms involved in the photodynamic action following PDT in human cell lines of poorly differentiated (CNE2) and moderately differentiated (TW0-1) nasopharyngeal carcinoma (NPC) and also poorly differentiated colon (CCL-220.1) and bladder (SD) cells.

Nitric oxide mediated photo-induced cell death in human malignant cells.

Photodynamic therapy (PDT) is a therapeutic modality used for the treatment of a variety of solid neoplasms. The principle of PDT is based on the selective uptake of a photosensitizing chemical in tumor tissue/cell followed by irradiation of tumors with visible light. The treatment results in a cascade of oxidative events causing cell death both in vitro and in vivo. Nitric oxide (NO) is a gaseous free radical, which is an important modulator of immune, endocrine and neuronal functions and plays an important role in the induction of apoptosis. Hypericin (HY) is a photosensitizing pigment from Hypericum perforatum that displays phototoxic effects in neoplastic cell lines. Our previous studies have shown HY induced apoptotic cell death in nasopharyngeal carcinoma and other tumor cells. To better understand the oxidative mechanism of apoptosis induced by HY, we hypothesized the role of NO in PDT, which is considered to be involved in a variety of physiological and pathological processes. We first demonstrated the presence of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) reactivity, a potential marker of NO synthesizing (NOS) enzyme both at light (LM) and electron microscopic (EM) level. Immunocytochemistry, using specific antibodies for NOS subtypes (constitutive, NOS I and inducible, NOS II), we observed that both NOS I and NOS II was present in all cell lines. The expression of both NOS I and NOS II was further verified using Western blot analysis as early as 15 min post PDT compared to that of drug-treated non-irradiated and light alone treated control cells. Our observation of NO production and distribution using the DAF-2 method is direct evidence of NO production in PDT-treated cells.

Bio-distribution and subcellular localization of Hypericin and its role in PDT induced apoptosis in cancer cells.

The development of new-generation photosensitizers to improve photodynamic therapy (PDT) and photodynamic diagnosis (PDD) is an area of extensive research. One such compound that has been studied in our group is Hypericin (HY). To study the mechanism of action we have investigated uptake, intracellular localization, cell phototoxicity and morphological changes especially to ultrastructures following photodynamic treatment in poorly (CNE2) and moderately (TW0-1) differentiated human nasopharyngeal carcinoma (NPC) cells and also other tumor cells such as colon (CCL-220.1) and bladder (SD) cells in vitro. Following irradiation, phototoxicity was determined by crystal fast violet assay and apoptosis was assessed using annexin-V assay. Using spectrofluorimetry and confocal laser scanning microscopy (CLSM) we have determined cellular fluorescence localization and uptake of HY. Co-labeling with HY and fluorescent dyes specific for cell organelles revealed an intracellular localization of HY predominantly in mitochondria and lysosomes. Since many photosensitizing agents in current clinical use have mitochondrial targets, HY may be a valuable addition to current protocols. In addition, our results also indicate that leakage of lysosomal protease into cytosolic compartment might be involved in the induction of apoptosis. Electron microscopy revealed damage to plasma membrane with high drug dose (>5 microM); indicating a mechanism related to necrosis, whereas sub-lethal lower doses (<2.5 microM) resulted in induction of apoptosis indicated by typical ultrastructural signs of apoptosis. Our results based on mitochondrial and lysosomal localization support the idea that PDT can contribute to elimination of malignant cells by the induction of apoptosis, and can be of physiological significance.

Efficacy of hypocrellin pharmacokinetics in phototherapy.

Hypocrellins A and B are pigments which are isolated from the parasitic fungi Hypocrella bambuase sacc and Shiraia bambusicola P. Heen found in the People's Republic of China and other parts of Asia including Sri Lanka. These agents, which belong to the general class of perylene quinonoid pigments, have a long history of traditional medicinal agents especially in Asia. Hypocrellins are under extensive investigation as photosensitizing agents for photodynamic therapy (PDT). Hypocrellin compounds were selected as potential photosensitizers for PDT owing to their high quantum yields of singlet oxygen (1O2), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. The cellular uptake, evaluated by spectrofluorimetry and confocal laser scanning microscopy (CLSM) demonstrated that both HA and HB exhibited high and fast uptake and rapid internalization as revealed by their bio-distribution pattern. In addition, the present study employed both immunocytochemical and Western blot techniques to explore the photo-induced expression of apoptosis related proteins in NPC as well as other human carcinoma cells. Using spectrofluorimetry and CLSM we have determined the cellular fluorescence as a marker for the uptake of HA and HB. Co-staining with either HA or HB and fluorescent dyes specific for cell organelles revealed an intracellular localization of HA and HB in lysosomes other than mitochondria.

Hypericin induced death receptor-mediated apoptosis in photoactivated tumor cells.

Nasopharyngeal carcinoma (NPC) is a malignant disease of the head/neck region with a 5-year survival level of approximately 65%. To explore the novel therapeutic strategies in the management of this disease, the potential effects of photodynamic therapy (PDT) in NPC cells were investigated. PDT, a new mode of treatment, is based on the combined use of light-absorbing compounds and light irradiation. Two human NPC cells such as, poorly differentiated (NPC/CNE2) and moderately differentiated (NPC/TW0-1) and other types of tumor cells like colon (CCL-220.1) and bladder (SD) undergo rapid apoptosis when treated with PDT sensitized with hypericin (HY). It has been shown that this compound has a strong photodynamic effect on tumors and viruses. However, the initiating events of PDT sensitized HY-induced apoptosis are not identified completely. In this study, we sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events are an early event in HY-treated PDT induced apoptosis. Loss of mitochondrial transmembrane potential, release of cytochrome c, involvement of caspases 8 and 3 and the status caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells. Photosensitization of HY enhanced both CD95/CD95L expression and induced CD95-signaling dependent cell death in all tumor cell lines studied. CD95/CD95L expression appeared within 2 h following light irradiation and appeared to be a principal event in PDT induced apoptosis. Furthermore, these results indicate that release of mitochondrial cytochrome c into the cytoplasm within 2-3 h post PDT is a secondary event following the activation of initiator caspase-8 preceding Apaf-1, caspase-9 and caspase-3 activation, cleavage of PARP and DNA fragmentation.

Photodynamic therapy induced Fas-mediated apoptosis in human carcinoma cells.

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. Human poorly (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells undergo rapid apoptosis when treated with PDT sensitized with Hypocrellin A (HA) and Hypocrellin B (HB). It has been shown that these compounds have a strong photodynamic effect on tumors and viruses. The initiating events of PDT sensitized HA and HB-induced apoptosis are poorly defined. In the current study, we sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events are an early event in HA and HB-treated PDT induced apoptosis. Loss of mitochondrial transmembrane potential, release of cytochrome c, involvement of caspases-8 and -3 and the status caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells. Photoactivation of HA and HB enhanced both CD95/CD95L expression and induced CD95-signaling dependent cell death in all tumor cell lines studied. CD95/ CD95L expression appeared within 2 h following light activation and appeared to be a primary event in PDT induced apoptosis. Furthermore, these results indicate that release of mitochondrial cytochrome c into the cytoplasm is a secondary event following the activation of initiator caspase-8 preceding caspase-3 activation, cleavage of PARP and DNA fragmentation. Cytochrome c appeared in the cytosol within 2-3 h post PDT. Cleavage of PARP was observed at 3-4 h following PDT and caspase-3 specific inhibitor DEVD-CHO and broad-spectrum caspases inhibitor z-VAD-fmk blocked caspase-3 activation and PARP cleavage suggesting that caspase-3 plays an important role in HA and HB-induced apoptosis.

Hypocrellins and Hypericin induced apoptosis in human tumor cells: a possible role of hydrogen peroxide.

We examined whether generation of H2O2 is a critical event for the apoptotic pathway upstream of mitochondrial involvement and caspase-3 protease activation. Perylquinone photosensitizers such as Hypocrellin A (HA), Hypocrellin B (HB) and Hypericin (HY) induced activation of caspase-3 and apoptosis upon photoactivation. Generation of H2O2 was commonly detected after photoactivation within an hour, and scavenging of H2O2 caused cells to fail to undergo apoptosis. Flow cytometry demonstrated that H2O2 production preceded loss of mitochondrial membrane potential (DeltaPsim) in photoactivated cells treated with HA, HB and HY. Then caspase-3 activity was activated, followed by DNA fragmentation. These findings suggest that HA, HB and HY upon photoactivation induce H2O2 generation, which causes (DeltaPsim) and subsequently caspase-3 activation, resulting in apoptosis. These findings suggest that generation of H2O2 by photoactivation of HA, HB and HY causes activation of caspase-3. Therefore, H2O2 may function as a common mediator for apoptosis induced by HA, HB and HY. The present study also demonstrated that upon photoactivation HA, HB and HY induced a decrease in intracellular acidification, glutathione (GSH) depletion and an array of mitochondrial damage together with apoptotic morphological changes in the irradiated cells.

Apoptosis induced by photosensitizers (Perylquinone derivatives) in human carcinoma cells: a possible relevance to photodynamic therapy.

Novel photosensitizers Hypocrellin A (HA) and Hypocrellin B (HB), lipid soluble perylquinone derivatives of the genus Hypericum have a strong photodynamic effect on tumors and viruses. However, the mechanisms of tumor cell death induced by HA and HB are still unclear. In this study, we attempt to elucidate the photodynamic effects of HA and HB compounds in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells as well as human mucosal colon (CCL-220.1) and bladder (SD) cells. Using these cell lines we investigated few hall marks of apoptotic commitments in a drug and light dose dependent manner. Tumor cells photoactivated with HA and HB showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by all tumor cell lines as evidenced by the externalization of phosphatidylserine. Western blot analysis of poly (ADP-ribose) polymerase, a caspases substrate, showed the classical cleavage pattern (116 to 85kDa) associated with apoptosis in HA and HB-treated cell lysates. In addition, PARP cleavage was blocked by using tetrapepdide caspases inhibitors such as DEVD or z-VAD. These results demonstrate that tumor cell death induced by HB and HA is mediated by caspase proteases. This study also identifies both colon and bladder cells were more sensitive cell lines than NPC (CNE2 and TWO-1) cell lines.

Photolon™ --photosensitization induces apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes.

The localization of photosensitizers in the subcellular compartments during photodynamic therapy (PDT) plays a major role in the cell destruction; therefore, the aim of this study was to investigate the intracellular localization of Chlorin e6-PVP (Photolon™) in malignant and normal cells. Our study involves the characterization of the structural determinants of subcellular localization of Photolon, and how subcellular localization affects the selective toxicity of Photolon towards tumor cells. Using confocal laser scanning microscopy (CLSM) and fluorescent organelle probes; we examined the subcellular localization of Photolon™ in the murine colon carcinoma CT-26 and normal fibroblast (NHLC) cells. Our results demonstrated that after 30 min of incubation, the distribution of Photolon was localized mainly in the cytoplasmic organelles including the mitochondria, lysosomes, Golgi apparatus, around the nuclear envelope and also in the nucleus but not in the endo-plasmic reticulum whereas in NHLC cells, Photolon was found to be localized minimally only in the nucleus not in other organelles studied. The relationship between subcellular localization of Photolon and PDT-induced apoptosis was investigated. Apoptotic cell death was judged by the formation of known apoptotic hallmarks including, the phosphatidylserine externalization (PS), PARP cleavage, a substrate for caspase-3 and the formation of apoptotic nuclei. At the irradiation dose of 1 J/cm2, the percentage of apoptotic cells was 80%, respectively. This study provided substantial evidence that Photolon preferentially localized in the subcellular organelles in the following order: nucleus, mitochondria, lysosomes and the Golgi apparatus and subsequent photodamage of the mitochondria and lyso-somes played an important role in PDT-mediated apoptosis CT-26 cells. Our results based on the cytoplasmic organelles and the intranuclear localization extensively enhance the efficacy of PDT with appropriate photosensitizer and light dose and support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.

Genome-wide promoter analysis of the SOX4 transcriptional network in prostate cancer cells.

SOX4 is a critical developmental transcription factor in vertebrates and is required for precise differentiation and proliferation in multiple tissues. In addition, SOX4 is overexpressed in many human malignancies, but the exact role of SOX4 in cancer progression is not well understood. Here, we have identified the direct transcriptional targets of SOX4 using a combination of genome-wide localization chromatin immunoprecipitation-chip analysis and transient overexpression followed by expression profiling in a prostate cancer model cell line. We have also used protein-binding microarrays to derive a novel SOX4-specific position-weight matrix and determined that SOX4 binding sites are enriched in SOX4-bound promoter regions. Direct transcriptional targets of SOX4 include several key cellular regulators, such as EGFR, HSP70, Tenascin C, Frizzled-5, Patched-1, and Delta-like 1. We also show that SOX4 targets 23 transcription factors, such as MLL, FOXA1, ZNF281, and NKX3-1. In addition, SOX4 directly regulates expression of three components of the RNA-induced silencing complex, namely Dicer, Argonaute 1, and RNA Helicase A. These data provide new insights into how SOX4 affects developmental signaling pathways and how these changes may influence cancer progression via regulation of gene networks involved in microRNA processing, transcriptional regulation, the TGFbeta, Wnt, Hedgehog, and Notch pathways, growth factor signaling, and tumor metastasis.

Nuclear-cytoplasmic transport of EGFR involves receptor endocytosis, importin beta1 and CRM1.

Many receptor tyrosine kinases (RTKs) can be detected in the cell nucleus, such as EGFR, HER-2, HER-3, HER-4, and fibroblast growth factor receptor. EGFR, HER-2 and HER-4 contain transactivational activity and function as transcription co-factors to activate gene promoters. High EGFR in tumor nuclei correlates with increased tumor proliferation and poor survival in cancer patients. However, the mechanism by which cell-surface EGFR translocates into the cell nucleus remains largely unknown. Here, we found that EGFR co-localizes and interacts with importins alpha1/beta1, carriers that are critical for macromolecules nuclear import. EGFR variant mutated at the nuclear localization signal (NLS) is defective in associating with importins and in entering the nuclei indicating that EGFR's NLS is critical for EGFR/importins interaction and EGFR nuclear import. Moreover, disruption of receptor internalization process using chemicals and forced expression of dominant-negative Dynamin II mutant suppressed nuclear entry of EGFR. Additional evidences suggest an involvement of endosomal sorting machinery in EGFR nuclear translocalization. Finally, we found that nuclear export of EGFR may involve CRM1 exportin as we detected EGFR/CRM1 interaction and markedly increased nuclear EGFR following exposure to leptomycin B, a CRM1 inhibitor. Collectively, these data suggest the importance of receptor endocytosis, endosomal sorting machinery, interaction with importins alpha1/beta1, and exportin CRM1 in EGFR nuclear-cytoplasmic trafficking. Together, our work sheds light into the nature and regulation of the nuclear EGFR pathway and provides a plausible mechanism by which cells shuttle cell-surface EGFR and potentially other RTKs through the nuclear pore complex and into the nuclear compartment.