Single Amino Acid Differences between Closely Related Reovirus T3D Lab Strains Alter Oncolytic Potency In Vitro and In Vivo.

Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.

Antitumor Benefits of Antiviral Immunity: An Underappreciated Aspect of Oncolytic Virotherapies.

Oncolytic viruses (OVs) represent a new class of cancer immunotherapeutics. Administration of OVs to cancer-bearing hosts induces two distinct immunities: antiviral and antitumor. While antitumor immunity is beneficial, antiviral immune responses are often considered detrimental for the efficacy of OV-based therapy. The existing dogma postulates that anti-OV immune responses restrict viral replication and spread, and thus reduce direct OV-mediated killing of cancer cells. Accordingly, a myriad of therapeutic strategies aimed at mitigating anti-OV immune responses is presently being tested. Here, we advocate that OV-induced antiviral immune responses hold intrinsic anticancer benefits and are essential for establishing clinically desired antitumor immunity. Thus, to achieve the optimal efficacy of OV-based cancer immunotherapies, strategic management of anti-OV immune responses is of critical importance.

MHC-I Ligand Discovery Using Targeted Database Searches of Mass Spectrometry Data: Implications for T-Cell Immunotherapies.

Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior to traditional "no enzyme" searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential.

Quantitative Temporal in Vivo Proteomics Deciphers the Transition of Virus-Driven Myeloid Cells into M2 Macrophages.

Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host-virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G-, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G-, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral-host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection.

Newly recruited CD11b+, GR-1+, Ly6C(high) myeloid cells augment tumor-associated immunosuppression immediately following the therapeutic administration of oncolytic reovirus.

Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b(+), Gr-1(+), Ly6C(high) myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b(+), Gr-1(+), Ly6C(high) myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b(+), Gr-1(+), Ly6C(high) myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus-based anticancer immunotherapies.

NAD+ salvage pathway in cancer metabolism and therapy.

Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme for various physiological processes including energy metabolism, DNA repair, cell growth, and cell death. Many of these pathways are typically dysregulated in cancer cells, making NAD+ an intriguing target for cancer therapeutics. NAD+ is mainly synthesized by the NAD+ salvage pathway in cancer cells, and not surprisingly, the pharmacological targeting of the NAD+ salvage pathway causes cancer cell cytotoxicity in vitro and in vivo. Several studies have described the precise consequences of NAD+ depletion on cancer biology, and have demonstrated that NAD+ depletion results in depletion of energy levels through lowered rates of glycolysis, reduced citric acid cycle activity, and decreased oxidative phosphorylation. Additionally, depletion of NAD+ causes sensitization of cancer cells to oxidative damage by disruption of the anti-oxidant defense system, decreased cell proliferation, and initiation of cell death through manipulation of cell signaling pathways (e.g., SIRT1 and p53). Recently, studies have explored the effect of well-known cancer therapeutics in combination with pharmacological depletion of NAD+ levels, and found in many cases a synergistic effect on cancer cell cytotoxicity. In this context, we will discuss the effects of NAD+ salvage pathway inhibition on cancer cell biology and provide insight on this pathway as a novel anti-cancer therapeutic target.

Multifaceted therapeutic targeting of ovarian peritoneal carcinomatosis through virus-induced immunomodulation.

Immunosuppression associated with ovarian cancer (OC) and resultant peritoneal carcinomatosis (PC) hampers the efficacy of many promising treatment options, including immunotherapies. It is hypothesized that oncolytic virus-based therapies can simultaneously kill OC and mitigate immunosuppression. Currently, reovirus-based anticancer therapy is undergoing phase I/II clinical trials for the treatment of OC. Hence, this study was focused on characterizing the effects of reovirus therapy on OC and associated immune microenvironment. Our data shows that reovirus efficiently killed OC cells and induced higher expression of the molecules involved in antigen presentation including major histocompatibility complex (MHC) class I, ß2-microglobulin (ß2M), TAP-1, and TAP-2. In addition, in the presence of reovirus, dendritic cells (DCs) overcame the OC-mediated phenotypic suppression and successfully stimulated tumor-specific CD8+ T cells. In animal studies, reovirus targeted local and distal OC, alleviated the severity of PC and significantly prolonged survival. These therapeutic effects were accompanied by decreased frequency of suppressive cells, e.g., Gr1.1+, CD11b+ myeloid derived suppressor cells (MDSCs), and CD4+, CD25+, FOXP3+ Tregs, tumor-infiltration of CD3+ cells and higher expression of Th1 cytokines. Finally, reovirus therapy during early stages of OC also resulted in the postponement of PC development. This report elucidates timely information on a therapeutic approach that can target OC through clinically desired multifaceted mechanisms to better the outcomes.

Reovirus variants with mutations in genome segments S1 and L2 exhibit enhanced virion infectivity and superior oncolysis.

Reovirus preferentially replicates in transformed cells and is being explored as a cancer therapy. Immunological and physical barriers to virotherapy inspired a quest for reovirus variants with enhanced oncolytic potency. Using a classical genetics approach, we isolated two reovirus variants (T3v1 and T3v2) with superior replication relative to wild-type reovirus serotype 3 Dearing (T3wt) on various human and mouse tumorigenic cell lines. Unique mutations in reovirus λ2 vertex protein and σ1 cell attachment protein were associated with the large plaque-forming phenotype of T3v1 and T3v2, respectively. Both T3v1 and T3v2 exhibited higher infectivity (i.e., a higher PFU-to-particle ratio) than T3wt. A detailed analysis of virus replication revealed that virus cell binding and uncoating were equivalent for variant and wild-type reoviruses. However, T3v1 and T3v2 were significantly more efficient than T3wt in initiating productive infection. Thus, when cells were infected with equivalent input virus particles, T3v1 and T3v2 produced significantly higher levels of early viral RNAs relative to T3wt. Subsequent steps of virus replication (viral RNA and protein synthesis, virus assembly, and cell death) were equivalent for all three viruses. In a syngeneic mouse model of melanoma, both T3v1 and T3v2 prolonged mouse survival compared to wild-type reovirus. Our studies reveal that oncolytic potency of reovirus can be improved through distinct mutations that increase the infectivity of reovirus particles.

Aldehyde dehydrogenase activity of breast cancer stem cells is primarily due to isoform ALDH1A3 and its expression is predictive of metastasis.

Cancer stem cells (CSCs) are proposed to initiate cancer and propagate metastasis. Breast CSCs identified by aldehyde dehydrogenase (ALDH) activity are highly tumorigenic in xenograft models. However, in patient breast tumor immunohistological studies, where CSCs are identified by expression of ALDH isoform ALDH1A1, CSC prevalence is not correlative with metastasis, raising some doubt as to the role of CSCs in cancer. We characterized the expression of all 19 ALDH isoforms in patient breast tumor CSCs and breast cancer cell lines by total genome microarray expression analysis, immunofluorescence protein expression studies, and quantitative polymerase chain reaction. These studies revealed that ALDH activity of patient breast tumor CSCs and cell lines correlates best with expression of another isoform, ALDH1A3, not ALDH1A1. We performed shRNA knockdown experiments of the various ALDH isoforms and found that only ALDH1A3 knockdown uniformly reduced ALDH activity of breast cancer cells. Immunohistological studies with fixed patient breast tumor samples revealed that ALDH1A3 expression in patient breast tumors correlates significantly with tumor grade, metastasis, and cancer stage. Our results, therefore, identify ALDH1A3 as a novel CSC marker with potential clinical prognostic applicability, and demonstrate a clear correlation between CSC prevalence and the development of metastatic breast cancer.

Oncolytic virus-initiated protective immunity against prostate cancer.

Recently reovirus-based oncotherapy has been successfully implemented for the treatment of prostate cancer. In this report, we show that apart from its primary direct cancer-killing activity, reovirus oncotherapy overrides tumor-associated immune evasion strategies and confers protective antiprostate cancer immunity. Prostate cancer represents an ideal target for immunotherapies. However, currently available immune interventions fail to induce clinically significant antiprostate cancer immune responses, owing to the immunosuppressive microenvironment associated with this disease. We show here that during the process of oncolysis, reovirus acts upon prostate cancer cells and initiates proinflammatory cytokines and major histocompatibility complex (MHC) class I molecule expression. In an immunocompetent transgenic adenocarcinoma of mouse prostate (TRAMP) model, reovirus oncotherapy induces the homing of CD8(+) T and NK cells in tumors and the display of tumor-associated antigens (TAAs) on antigen-presenting cells (APCs), and endows dendritic cells (DCs) with a capacity to successfully present TAAs to tumor-specific CD8(+) T cells. These newly generated immunological events lead to the development of strong antiprostate cancer T cell responses, which restrict the growth of subsequently, implanted syngeneic tumor in an antigen-specific, but reovirus-independent manner. Such reovirus-initiated antiprostate cancer immunity represents a clinically valuable entity that can promote long-term cancer-free health even after discontinuation of the primary oncotherapy.

Curcumin-induced apoptosis in ovarian carcinoma cells is p53-independent and involves p38 mitogen-activated protein kinase activation and downregulation of Bcl-2 and survivin expression and Akt signaling.

New cytotoxic agents are urgently needed for the treatment of advanced ovarian cancer because of the poor long-term response of this disease to conventional chemotherapy. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity; however, the mechanism of curcumin-induced cytotoxicity in ovarian cancer cells remains a mystery. In this study we show that curcumin exhibited time- and dose-dependent cytotoxicity against monolayer cultures of ovarian carcinoma cell lines with differing p53 status (wild-type p53: HEY, OVCA429; mutant p53: OCC1; null p53: SKOV3). In addition, p53 knockdown or p53 inhibition did not diminish curcumin killing of HEY cells, confirming p53-independent cytotoxicity. Curcumin also killed OVCA429, and SKOV3 cells grown as multicellular spheroids. Nuclear condensation and fragmentation, as well as DNA fragmentation and poly (ADP-ribose) polymerase-1 cleavage in curcumin-treated HEY cells, indicated cell death by apoptosis. Procaspase-3, procaspase-8, and procaspase-9 cleavage, in addition to cytochrome c release and Bid cleavage into truncated Bid, revealed that curcumin activated both the extrinsic and intrinsic pathways of apoptosis. Bax expression was unchanged but Bcl-2, survivin, phosphorylated Akt (on serine 473), and total Akt were downregulated in curcumin-treated HEY cells. Curcumin also activated p38 mitogen-activated protein kinase (MAPK) without altering extracellular signal-regulated kinase 1/2 activity. We conclude that p53-independent curcumin-induced apoptosis in ovarian carcinoma cells involves p38 MAPK activation, ablation of prosurvival Akt signaling, and reduced expression of the antiapoptotic proteins Bcl-2 and survivin. These data provide a mechanistic rationale for the potential use of curcumin in the treatment of ovarian cancer.

Human mast cell activation with virus-associated stimuli leads to the selective chemotaxis of natural killer cells by a CXCL8-dependent mechanism.

Human mast cells are found in skin and mucosal surfaces and next to blood vessels. They play a sentinel cell role in immunity, recognizing invading pathogens and producing proinflammatory mediators. Mast cells can recruit granulocytes, and monocytes in allergic disease and bacterial infection, but their ability to recruit antiviral effector cells such as natural killer (NK) cells and T cells has not been fully elucidated. To investigate the role of human mast cells in response to virus-associated stimuli, human cord blood-derived mast cells (CBMCs) were stimulated with polyinosinic.polycytidylic acid, a double-stranded RNA analog, or infected with the double-stranded RNA virus, reovirus serotype 3 Dearing for 24 hours. CBMCs responded to stimulation with polyinosinic.polycytidylic acid by producing a distinct chemokine profile, including CCL4, CXCL8, and CXCL10. CBMCs produced significant amounts of CXCL8 in response to low levels of reovirus infection, while both skin- and lung-derived fibroblasts were unresponsive unless higher doses of reovirus were used. Supernatants from CBMCs infected with reovirus induced substantial NK cell chemotaxis that was highly dependent on CXCL8 and CXCR1. These results suggest a novel role for mast cells in the recruitment of human NK cells to sites of early viral infection via CXCL8.

Oncolytic reovirus effectively targets breast cancer stem cells.

Recent evidence suggests that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are resistant to current anticancer therapies. Hence, novel cancer therapies will need to be tested for both tumor regression and CSC targeting. Herein we show that oncolytic reovirus that induces regression of human breast cancer primary tumor samples xenografted in immunocompromised mice also effectively targets and kills CSCs in these tumors. CSCs were identified based on CD24(-)CD44(+) cell surface expression and overexpression of aldehyde dehydrogenase. Upon reovirus treatment, the CSC population was reduced at the same rate as non-CSCs within the tumor. Immunofluorescence of breast tumor tissue samples from the reovirus- and mock-treated mice confirmed that both CSCs and non-CSCs were infectible by reovirus, and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay showed that both populations died by apoptosis. Ras, which has been shown to mediate reovirus oncolysis, was found to be present at similar levels in all cell types, and this is consistent with their comparable sensitivity to reovirus. These experiments indicate that oncolytic reovirus has the potential to induce tumor regression in breast cancer patients. More important, the CSC population was equally reduced and was as susceptible to reovirus treatment as the non-CSC population.

Chromium-mediated apoptosis: involvement of DNA-dependent protein kinase (DNA-PK) and differential induction of p53 target genes.

Cellular stress and DNA damage up-regulate and activate p53, fundamental for cell cycle control, senescence, DNA repair and apoptosis. The specific mechanism(s) that determine whether p53-dependent cell cycle arrest or p53-dependent apoptosis prevails in response to specific DNA damage are poorly understood. In this study, we investigated two types of DNA damage, chromium treatment and gamma irradiation (IR) that induced similar levels of p53, but that mediated two distinct p53-dependent cell fates. Chromium exposure induced a robust DNA-dependent protein kinase (DNA-PK)-mediated apoptotic response that was accompanied by the rapid loss of the cyclin-dependent kinase inhibitor 1A (p21) protein, whereas IR treatment-induced cell cycle arrests that was supported by the rapid induction of p21. Inhibition of DNA-PK effectively blocked chromium-, but not IR-induced p53 stabilization and activation. In contrast, inhibition of ATM and ATR by caffeine had the inverse effect of blocking IR-, but not chromium-induced p53 stabilization and activation. Chromium exposure ablated p21 transcription but PUMA and Bax transcription was significantly enhanced compared to non-damaged cells. In contrast, IR treatment triggered significant p21 mRNA synthesis in addition to PUMA and Bax mRNA production. While chromium treatment enhanced the binding of p53 and RNA polymerase II (RNA Pol II) to both the p21 and PUMA promoters, RNA Pol II elongation was only observed along the PUMA gene and not the p21 gene. In contrast, following IR treatment, RNA Pol II elongation was observed on both p21 and PUMA. Chromium-induced apoptosis therefore involves DNA-PK-mediated p53 activation followed by preferential transcription of pro-apoptotic PUMA over anti-apoptotic p21 genes.

Hypersensitivity to chromium-induced DNA damage correlates with constitutive deregulation of upstream p53 kinases in p21-/- HCT116 colon cancer cells.

The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) is a key component in cell cycle control and apoptosis, directing an anti-apoptotic response following DNA damage. Chromium exposure resulted in a 500-1000 fold increase in apoptosis-induced cell death in p21-/- HCT116 cells compared to wild-type or p53-/- cells. p53 shRNA (or transient p53 siRNA) into p21-/- HCT116 cells reduced Cr(VI) sensitivity, suggesting the enhanced apoptosis in p21-/- cells is p53-dependent. Under non-DNA damage conditions, the p53 level in p21-/- cells was significantly higher than in wild-type cells, due to enhanced p53 phosphorylation and stabilization rather than elevated p53 transcription. Wild-type cells showed significant p53 protein induction upon DNA damage whereas p21-/- cells showed no p53 increase. p21-/- cells display the constitutive activation of upstream p53 kinases (ATM, DNA-PK, ATR, AKT and p38). 2D gel analysis revealed p53 patterns in p21-/- cells were distinct from those in wild-type cells before and after chromium exposure. Our results suggest that p21 has an important role in the cellular response to normal replicative stress and its absence leads to a "chronic DNA damage" state that primes the cell for p53-dependent apoptosis.

Public awareness and use of direct-to-consumer genetic tests: results from 3 state population-based surveys, 2006.

We conducted population-based surveys on direct-to-consumer nutrigenomic testing in Michigan, Oregon, and Utah as part of the 2006 Behavioral Risk Factor Surveillance System. Awareness of the tests was highest in Oregon (24.4%) and lowest in Michigan (7.6%). Predictors of awareness were more education, higher income, and increasing age, except among those 65 years or older. Less than 1% had used a health-related direct-to-consumer genetic test. Public health systems should increase consumer and provider education and continue surveillance on direct-to-consumer genetic tests.

Retraction: DNA-PKCS binding to p53 on the p21WAF1/CIP1 promoter blocks transcription resulting in cell death.

[This retracts the article DOI: 10.18632/oncotarget.378.].

Regulation of Cancer and Cancer-Related Genes via NAD.

SIGNIFICANCE: NAD+ is an essential redox cofactor in cellular metabolism and has emerged as an important regulator of a wide spectrum of disease conditions, most notably, cancers. As such, various strategies targeting NAD+ synthesis in cancers are in clinical trials. Recent Advances: Being a substrate required for the activity of various enzyme families, especially sirtuins and poly(adenosine diphosphate [ADP]-ribose) polymerases, NAD+-mediated signaling plays an important role in gene expression, calcium release, cell cycle progression, DNA repair, and cell proliferation. Many strategies exploring the potential of interfering with NAD+ metabolism to sensitize cancer cells to achieve anticancer benefits are highly promising, and are being pursued. CRITICAL ISSUES: With the multifaceted roles of NAD+ in cancer, it is important to understand how cellular processes are reliant on NAD+. This review summarizes how NAD+ metabolism regulates various pathophysiological processes in cancer, and how this knowledge can be exploited to devise effective anticancer therapies in clinical settings. FUTURE DIRECTIONS: In line with the redundant pathways that facilitate NAD+ metabolism, further studies should comprehensively understand the roles of the various NAD+-synthesizing as well as NAD+-utilizing biomolecules to understand its true potential in cancer treatment.

HDAC6 differentially regulates autophagy in stem-like versus differentiated cancer cells.

Cancer stem-like cells (CSCs), a small population of pluripotent cells residing within heterogeneous tumor mass, remain highly resistant to various chemotherapies as compared to the differentiated cancer cells. It is being postulated that CSCs possess unique molecular mechanisms, such as autophagic homeostasis, that allow CSCs to withstand the therapeutic assaults. Here we demonstrate that HDAC6 inhibition differentially modulates macroautophagy/autophagy in CSCs as compared to that of differentiated cancer cells. Using human and murine CSC models and differentiated cells, we show that the inhibition or knockdown (KD) of HDAC6 decreases CSC pluripotency by downregulating major pluripotency factors POU5F1, NANOG and SOX2. This decreased HDAC6 expression increases ACTB, TUBB3 and CSN2 expression and promotes differentiation in CSCs in an apoptosis-independent manner. Mechanistically, HDAC6 KD in CSCs decreases pluripotency by promoting autophagy, whereas the inhibition of pluripotency via retinoic acid treatment, POU5F1 or autophagy-related gene (ATG7 and ATG12) KD in CSCs decreases HDAC6 expression and promotes differentiation. Interestingly, HDAC6 KD-mediated CSC growth inhibition is further enhanced in the presence of autophagy inducers Tat-Beclin 1 peptide and rapamycin. In contrast to the results observed in CSCs, HDAC6 KD in differentiated breast cancer cells downregulates autophagy and increases apoptosis. Furthermore, the autophagy regulator p-MTOR, upstream negative regulators of p-MTOR (TSC1 and TSC2) and downstream effectors of p-MTOR (p-RPS6KB and p-EIF4EBP1) are differentially regulated in CSCs versus differentiated cancer cells following HDAC6 KD. Overall these data identify the differential regulation of autophagy as a molecular link behind the differing chemo-susceptibility of CSCs and differentiated cancer cells.

Curcumin induces apoptosis in HCT-116 human colon cancer cells in a p21-independent manner.

Several micronutrients present in fruits and vegetables exhibit anticancer activity as a result of their actions on molecular targets involved in carcinogenesis and tumor progression. Curcumin, a phenolic phytochemical derived from the rhizome of Curcuma longa, exhibits both cancer-preventative activity and growth inhibitory effects on neoplastic cells. Several studies report that curcumin inhibits cancer cell proliferation and induces apoptosis in cancer cells through p21-mediated cell cycle arrest. Cancer cells that are deficient in p21 are also reported to be more prone to undergo apoptosis in response to a variety of cytotoxic agents. In this study, we determined whether curcumin-induced cytotoxicity in cultures of HCT-116 human colon cancer cells was dependent on p21 status. Curcumin killed wild-type HCT-116 cells in a dose- and time-dependent manner, as measured in an MTT cell viability assay. Moreover, an equivalent cytotoxic effect by curcumin was observed in both p21(+/+) and p21(-/-)HCT-116 cells, indicating that curcumin-induced cytotoxicity was p21-independent. Primary cultures of human dermal fibroblasts were less sensitive than HCT-116 colon cancer cells to lower doses of curcumin, suggesting a degree of selectivity for neoplastic cells. Western blot analysis showed that cell death in curcumin-treated cultures of p21(+/+) and p21(-/-) HCT-116 cells was associated with a reduction in pro-caspase-3 and PARP-1 cleavage, which are indicative of apoptosis. We conclude that curcumin-induced apoptosis in HCT-116 colon cancer cells does not depend on p21 status.