Autophagy in stromal fibroblasts promotes tumor desmoplasia and mammary tumorigenesis.

Autophagy inhibitors are currently being evaluated in clinical trials for the treatment of diverse cancers, largely due to their ability to impede tumor cell survival and metabolic adaptation. More recently, there is growing interest in whether and how modulating autophagy in the host stroma influences tumorigenesis. Fibroblasts play prominent roles in cancer initiation and progression, including depositing type 1 collagen and other extracellular matrix (ECM) components, thereby stiffening the surrounding tissue to enhance tumor cell proliferation and survival, as well as secreting cytokines that modulate angiogenesis and the immune microenvironment. This constellation of phenotypes, pathologically termed desmoplasia, heralds poor prognosis and reduces patient survival. Using mouse mammary cancer models and syngeneic transplantation assays, we demonstrate that genetic ablation of stromal fibroblast autophagy significantly impedes fundamental elements of the stromal desmoplastic response, including collagen and proinflammatory cytokine secretion, extracellular matrix stiffening, and neoangiogenesis. As a result, autophagy in stromal fibroblasts is required for mammary tumor growth in vivo, even when the cancer cells themselves remain autophagy-competent . We propose the efficacy of autophagy inhibition is shaped by this ability of host stromal fibroblast autophagy to support tumor desmoplasia.

Autophagy-Dependent Shuttling of TBC1D5 Controls Plasma Membrane Translocation of GLUT1 and Glucose Uptake.

Autophagy traditionally sustains metabolism in stressed cells by promoting intracellular catabolism and nutrient recycling. Here, we demonstrate that in response to stresses requiring increased glycolytic demand, the core autophagy machinery also facilitates glucose uptake and glycolytic flux by promoting cell surface expression of the glucose transporter GLUT1/Slc2a1. During metabolic stress, LC3+ autophagic compartments bind and sequester the RabGAP protein TBC1D5 away from its inhibitory interactions with the retromer complex, thereby enabling retromer recruitment to endosome membranes and GLUT1 plasma membrane translocation. In contrast, TBC1D5 inhibitory interactions with the retromer are maintained in autophagy-deficient cells, leading to GLUT1 mis-sorting into endolysosomal compartments. Furthermore, TBC1D5 depletion in autophagy-deficient cells rescues retromer recruitment to endosomal membranes and GLUT1 surface recycling. Hence, TBC1D5 shuttling to autophagosomes during metabolic stress facilitates retromer-dependent GLUT1 trafficking. Overall, our results illuminate key interconnections between the autophagy and endosomal pathways dictating GLUT1 trafficking and extracellular nutrient uptake.

Role of p53 in silibinin-mediated inhibition of ultraviolet B radiation-induced DNA damage, inflammation and skin carcinogenesis.

Non-melanoma skin cancers (NMSC) are a growing problem given that solar ultraviolet B (UVB) radiation exposure is increasing most likely due to depletion of the atmospheric ozone layer and lack of adequate sun protection. Better preventive methods are urgently required to reduce UV-caused photodamage and NMSC incidence. Earlier, we have reported that silibinin treatment activates p53 and reduces photodamage and NMSC, both in vitro and in vivo; but whether silibinin exerts its protective effects primarily through p53 remains unknown. To address this question, we generated p53 heterozygous (p53+/-) and p53 knockout (p53-/-) mice on SKH-1 hairless mouse background, and assessed silibinin efficacy in both short- and long-term UVB exposure experiments. In the chronic UVB-exposed skin tumorigenesis study, compared to p53+/+ mice, p53+/- mice developed skin tumors earlier and had higher tumor number, multiplicity and volume. Silibinin topical treatment significantly reduced the tumor number, multiplicity and volume in p53+/+ mice but silibinin' protective efficacy was significantly compromised in p53+/- mice. Additionally, silibinin treatment failed to inhibit precursor skin cancer lesions in p53-/- mice but improved the survival of the mice. In short-term studies, silibinin application accelerated the removal of UVB-induced DNA damage in p53+/+ mice while its efficacy was partially compromised in p53-/- mice. Interestingly, silibinin treatment also inhibited the UVB-induced inflammatory markers in skin tissue. These results further confirmed that absence of the p53 allele predisposes mice to photodamage and photocarcinogenesis, and established that silibinin mediates its protection against UVB-induced photodamage, inflammation and photocarcinogenesis partly through p53 activation.

Absence of a p53 allele delays nitrogen mustard-induced early apoptosis and inflammation of murine skin.

Bifunctional alkylating agent sulfur mustard (SM) and its analog nitrogen mustard (NM) cause DNA damage leading to cell death, and potentially activating inflammation. Transcription factor p53 plays a critical role in DNA damage by regulating cell cycle progression and apoptosis. Earlier studies by our laboratory demonstrated phosphorylation of p53 at Ser15 and an increase in total p53 in epidermal cells both in vitro and in vivo following NM exposure. To elucidate the role of p53 in NM-induced skin toxicity, we employed SKH-1 hairless mice harboring wild type (WT) or heterozygous p53 (p53+/-). Exposure to NM (3.2mg) caused a more profound increase in epidermal thickness and apoptotic cell death in WT relative to p53+/- mice at 24h. However, by 72h after exposure, there was a comparable increase in NM-induced epidermal cell death in both WT and p53+/- mice. Myeloperoxidase activity data showed that neutrophil infiltration was strongly enhanced in NM-exposed WT mice at 24h persisting through 72h of exposure. Conversely, robust NM-induced neutrophil infiltration (comparable to WT mice) was seen only at 72h after exposure in p53+/- mice. Similarly, NM-exposure strongly induced macrophage and mast cell infiltration in WT, but not p53+/- mice. Together, these data indicate that early apoptosis and inflammation induced by NM in mouse skin are p53-dependent. Thus, targeting this pathway could be a novel strategy for developing countermeasures against vesicants-induced skin injury.

Silibinin prevents ultraviolet B radiation-induced epidermal damages in JB6 cells and mouse skin in a p53-GADD45α-dependent manner.

Better preventive strategies are required to reduce ultraviolet (UV)-caused photodamage, the primary etiological factor for non-melanoma skin cancer (NMSC). Accordingly, here we examined the preventive efficacy of silibinin against UVB-induced photodamage using mouse epidermal JB6 cells and SKH1 hairless mouse epidermis. In JB6 cells, silibinin pretreatment protected against apoptosis and accelerated the repair of cyclobutane pyrimidine dimers (CPD) induced by moderate dose of UVB (50 mJ/cm(2)), which we are at risk of daily exposure. Silibinin also reversed UVB-induced S phase arrest, reducing both active DNA synthesizing and inactive S phase populations. In mechanistic studies, UVB-irradiated cells showed a transient upregulation of both phosphorylated (Ser-15 and Ser-392) and total p53, whereas silibinin pretreatment led to a more sustained upregulation and stronger nuclear localization of p53. Silibinin also caused a marked upregulation of GADD45α, a downstream target of p53, implicated in DNA repair and cell cycle regulation. Importantly, under p53 and GADD45α knockdown conditions, cells were more susceptible to UVB-induced apoptosis without any significant S phase arrest, and protective effects of silibinin were compromised. Similar to the in vitro results, topical application of silibinin prior to or immediately after UVB irradiation resulted in sustained increase in p53 and GADD45α levels and accelerated CPD removal in the epidermis of SKH1 hairless mice. Together, our results show for the first time that p53-mediated GADD45α upregulation is the key mechanism by which silibinin protects against UVB-induced photodamage and provides a strong rationale to investigate silibinin in reducing the risk and/or preventing early onset of NMSC.

Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation.

The protumorigenic functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to diverse stresses. Here we demonstrate an unexpected connection between autophagy and glucose metabolism that facilitates adhesion-independent transformation driven by a strong oncogenic insult-mutationally active Ras. In cells ectopically expressing oncogenic H-Ras as well as human cancer cell lines harboring endogenous K-Ras mutations, autophagy is induced following extracellular matrix detachment. Inhibiting autophagy due to the genetic deletion or RNA interference-mediated depletion of multiple autophagy regulators attenuates Ras-mediated adhesion-independent transformation and proliferation as well as reduces glycolytic capacity. Furthermore, in contrast to autophagy-competent cells, both proliferation and transformation in autophagy-deficient cells expressing oncogenic Ras are insensitive to reductions in glucose availability. Overall, increased glycolysis in autophagy-competent cells facilitates Ras-mediated adhesion-independent transformation, suggesting a unique mechanism by which autophagy may promote Ras-driven tumor growth in specific metabolic contexts.

ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death.

ATG12, an ubiquitin-like modifier required for macroautophagy, has a single known conjugation target, another autophagy regulator called ATG5. Here, we identify ATG3 as a substrate for ATG12 conjugation. ATG3 is the E2-like enzyme necessary for ATG8/LC3 lipidation during autophagy. ATG12-ATG3 complex formation requires ATG7 as the E1 enzyme and ATG3 autocatalytic activity as the E2, resulting in the covalent linkage of ATG12 onto a single lysine on ATG3. Surprisingly, disrupting ATG12 conjugation to ATG3 does not affect starvation-induced autophagy. Rather, the lack of ATG12-ATG3 complex formation produces an expansion in mitochondrial mass and inhibits cell death mediated by mitochondrial pathways. Overall, these results unveil a role for ATG12-ATG3 in mitochondrial homeostasis and implicate the ATG12 conjugation system in cellular functions distinct from the early steps of autophagosome formation.

Autophagy inhibition and antimalarials promote cell death in gastrointestinal stromal tumor (GIST).

Although gastrointestinal stromal tumors (GISTs) harboring activating KIT or platelet-derived growth factor receptor A (PDGFRA) mutations respond to treatment with targeted KIT/PDGFRA inhibitors such as imatinib mesylate, these treatments are rarely curative. Most often, a sizeable tumor cell subpopulation survives and remains quiescent for years, eventually resulting in acquired resistance and treatment failure. Here, we report that imatinib induces autophagy as a survival pathway in quiescent GIST cells. Inhibiting autophagy, using RNAi-mediated silencing of autophagy regulators (ATGs) or antimalarial lysosomotrophic agents, promotes the death of GIST cells both in vitro and in vivo. Thus, combining imatinib with autophagy inhibition represents a potentially valuable strategy to promote GIST cytotoxicity and to diminish both cellular quiescence and acquired resistance in GIST patients.

Autophagy and tumorigenesis.

Autophagy, a catabolic process involved in the sequestration and lysosomal degradation of cytoplasmic contents, is crucial for cellular homeostasis. The current literature supports that autophagy plays diverse roles in the development, maintenance, and progression of tumors. While genetic evidence indicates autophagy functions as a tumor suppressor mechanism, it is also apparent that autophagy can promote the survival of established tumors under stress conditions and in response to chemotherapy. In this review, we discuss the mechanisms and the evidence underlying these multifaceted roles of autophagy in tumorigenesis, the prospects for targeting autophagy in cancer therapy, and overview the potential markers that may be utilized to reliably detect autophagy in clinical settings.

Inositol hexaphosphate suppresses growth and induces apoptosis in prostate carcinoma cells in culture and nude mouse xenograft: PI3K-Akt pathway as potential target.

Constitutive activation of phosphoinositide 3-kinase (PI3K)-Akt pathway transmits growth-regulatory signals that play a central role in promoting survival, proliferation, and angiogenesis in human prostate cancer cells. Here, we assessed the efficacy of inositol hexaphosphate (IP6) against invasive human prostate cancer PC-3 and C4-2B cells and regulation of PI3K-Akt pathway. IP6 treatment of cells suppressed proliferation, induced apoptosis along with caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, and inhibited constitutive activation of Akt and its upstream regulators PI3K, phosphoinositide-dependent kinase-1 and integrin-linked kinase-1 (ILK1). Downstream of Akt, IP6 inhibited the phosphorylation of glycogen synthase kinase-3alpha/beta at Ser(21/9) and consequently reduced cyclin D1 expression. Efficacy studies employing PC-3 tumor xenograft growth in nude mice showed that 2% (w/v) IP6 feeding in drinking water inhibits tumor growth and weight by 52% to 59% (P < 0.001). Immunohistochemical analysis of xenografts showed that IP6 significantly reduces the expression of molecules associated with cell survival/proliferation (ILK1, phosphorylated Akt, cyclin D1, and proliferating cell nuclear antigen) and angiogenesis (platelet endothelial cell adhesion molecule-1 or CD31, vascular endothelial growth factor, endothelial nitric oxide synthase, and hypoxia-inducible factor-1alpha) together with an increase in apoptotic markers (cleaved caspase-3 and PARP). These findings suggest that, by targeting the PI3K-ILK1-Akt pathway, IP6 suppresses cell survival, proliferation, and angiogenesis but induces death in prostate cancer cells, which might have translational potential in preventing and controlling the growth of advanced and aggressive prostate cancer for which conventional chemotherapy is not effective.

p21/Cip1 and p27/Kip1 Are essential molecular targets of inositol hexaphosphate for its antitumor efficacy against prostate cancer.

Inositol hexaphosphate (IP6) causes G(1) arrest and increases cyclin-dependent kinase inhibitors p21/Cip1 and p27/Kip1 protein levels in human prostate cancer (PCa) DU145 cells lacking functional p53. However, whether cyclin-dependent kinase inhibitor I induction by IP6 plays any role in its antitumor efficacy is unknown. Herein, we observed that either p21 or p27 knockdown by small interfering RNA has no considerable effect on IP6-induced G(1) arrest, growth inhibition, and death in DU145 cells; however, the simultaneous knockdown of both p21 and p27 reversed the effects of IP6. To further confirm these findings both in vitro and in vivo, we generated DU145 cell variants with knockdown levels of p21 (DU-p21), p27 (DU-p27), or both (DU-p21+p27) via retroviral transduction of respective short hairpin RNAs. Knocking down p21 or p27 individually did not alter IP6-caused cell growth inhibition and G(1) arrest; however, their simultaneous ablation completely reversed the effects of IP6. In tumor xenograft studies, IP6 (2% w/v, in drinking water) caused a comparable reduction in tumor volume (40-46%) and tumor cell proliferation (26-28%) in DU-EV (control), DU-p21, and DU-p27 tumors but lost most of its effect in DU-p21+p27 tumors. IP6-caused apoptosis also occurred in a Cip/Kip-dependent manner because DU-p21+p27 cells were completely resistant to IP6-induced apoptosis both in cell culture and xenograft. Together, these results provide evidence, for the first time, of the critical role of p21 and p27 in mediating the anticancer efficacy of IP6, and suggest their redundant role in the antiproliferative and proapoptotic effects of IP6 in p53-lacking human PCa cells, both in vitro and in vivo.

Downregulation of both p21/Cip1 and p27/Kip1 produces a more aggressive prostate cancer phenotype.

Roles of cyclin dependent kinase inhibitors, p21/Cip1 (p21) and p27/Kip1 (p27) in prostate cancer (PCa) progression is still not clear. Lower p27 protein expression in PCa tissues is often associated with poor prognosis, but prognostic significance of p21 is still controversial. Herein, we investigated the role of these molecules in determining PCa growth characteristics. We generated human PCa DU145 cell variants with knocked down levels of p21 (DU-p21) or p27 (DU-p27), or both (DUp21 + p27) via retroviral transduction of respective shRNAs and compared their various characteristics with empty vector-transduced DU145 (DU-EV) cells in vitro as well as in vivo. Knocking down either p21 or p27 did not show any significant change in doubling time, clonogenicity and cell cycle progression in DU145 cells, but simultaneous knock-down of both p21 and p27 significantly enhanced these parameters. In athymic mice, DU-p21 + p27 tumors showed higher growth rate than the comparable growth of DU-EV, DU-p21 and DU-p27 tumors. Concurrently, DU-p21 + p27 tumors had significantly higher proliferation rate, showing 54% and 48% increase in proliferating cell nuclear antigen (PCNA) and Ki-67-positive cells, respectively, compared to DU-EV tumors. DU-p21 + p27 tumors also showed higher microvessel density and increased expression of vascular endothelial growth factor (VEGF). Proliferation and angiogenic status of DU-p21 and DU-p27 tumors was comparable to DU-EV tumors. Both in vitro and in vivo results implicate that p21 and p27 have compensatory roles in advanced prostate cancer cells, and ablation or downmodulation of both these molecules essentially enhances the aggressive prostate carcinoma phenotype.

p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells.

Recent studies have shown that silibinin induces p21/Cip1 and p27/Kip1 and G1 arrest in different prostate cancer cells irrespective of p53 status; however, biological significance and mechanism of such induction have not been studied. Here, using two different prostate cancer cell lines DU145 and 22Rv1, representing androgen-independent and androgen-dependent stages of malignancy, first we investigated the importance of p21 and p27 induction in silibinin-mediated G1 arrest. Silencing p21 and p27 individually by RNA interference showed marked reversal in G1 arrest; however, their simultaneous ablation showed additional reversal of G1 arrest in 22Rv1 but not DU145 cells. These results suggest that whereas relative importance of these molecules might be cell line specific, their induction by silibinin is essential for its G1 arrest effect. Next, studies were done to examine mechanisms of their induction where cycloheximide-chase experiments showed that silibinin increases p21 and p27 protein half-life. This effect was accompanied by strong reduction in Skp2 level and its binding with p21 and p27 together with strong decrease in phosphorylated Thr(187) p27 without considerable change in proteasomal activity, suggesting a posttranslational mechanism. Skp2 role was further elucidated using Skp2-small interfering RNA-transfected cells, where decreased G1 arrest and attenuated Cip/Kip induction were observed with silibinin treatment. Further, silibinin caused a marked increase in p21 and p27 mRNA levels together with an increase in their promoter activity, also indicating a transcriptional mechanism. Together, our results for the first time identify a central role of p21 and p27 induction and their regulatory mechanism in silibinin-mediated cell cycle arrest.