Sachin K. Deshmukh

p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling

Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24+/CD44+/EpCAM+) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24−/CD44−/EpCAM−) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemo-sensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC.

Comparative analysis of the relative potential of silver, Zinc-oxide and titanium-dioxide nanoparticles against UVB-induced DNA damage for the prevention of skin carcinogenesis

Sunscreen formulations containing UVB filters, such as Zinc-oxide (ZnO) and titanium-dioxide (TiO2) nanoparticles (NPs) have been developed to limit the exposure of human skin to UV-radiations. Unfortunately, these UVB protective agents have failed in controlling the skin cancer incidence. We recently demonstrated that silver nanoparticles (Ag-NPs) could serve as novel protective agents against UVB-radiations. Here our goal was to perform comparative analysis of direct and indirect UVB-protection efficacy of ZnO-, TiO2- and Ag-NPs. Sun-protection-factor calculated based on their UVB-reflective/absorption abilities was the highest for TiO2-NPs followed by Ag- and ZnO-NPs. This was further confirmed by studying indirect protection of UVB radiation-induced death of HaCaT cells. However, only Ag-NPs were active in protecting HaCaT cells against direct UVB-induced DNA-damage by repairing bulky-DNA lesions through nucleotide-excision-repair mechanism. Moreover, Ag-NPs were also effective in protecting HaCaT cells from UVB-induced oxidative DNA damage by enhancing SOD/CAT/GPx activity. In contrast, ZnO- and TiO2-NPs not only failed in providing any direct protection from DNA-damage, but rather enhanced oxidative DNA-damage by increasing ROS production. Together, these findings raise concerns about safety of ZnO- and TiO2-NPs and establish superior protective efficacy of Ag-NPs.

Development and Characterization of a Novel in vitro Progression Model for UVB-Induced Skin Carcinogenesis

Epidemiological studies suggest ultraviolet B (UVB) component (290–320 nm) of sun light is the most prevalent etiologic factor for skin carcinogenesis- a disease accounting for more than two million new cases each year in the USA alone. Development of UVB-induced skin carcinoma is a multistep and complex process. The molecular events that occur during UVB-induced skin carcinogenesis are poorly understood largely due to the lack of an appropriate cellular model system. Therefore, to make a progress in this area, we have developed an in vitro model for UVB-induced skin cancer using immortalized human epidermal keratinocyte (HaCaT) cells through repetitive exposure to UVB radiation. We demonstrate that UVB-transformed HaCaT cells gain enhanced proliferation rate, apoptosis-resistance, and colony- and sphere-forming abilities in a progressive manner. Moreover, these cells exhibit increased aggressiveness with enhanced migration and invasive potential and mesenchymal phenotypes. Furthermore, these derived cells are able to form aggressive squamous cell carcinoma upon inoculation into the nude mice, while parental HaCaT cells remain non-tumorigenic. Together, these novel, UVB-transformed progression model cell lines can be very helpful in gaining valuable mechanistic insight into UVB-induced skin carcinogenesis, identification of novel molecular targets of diagnostic and therapeutic significance, and in vitro screening for novel preventive and therapeutic agents.

Resistin potentiates chemoresistance and stemness of breast cancer cells: Implications for racially disparate therapeutic outcomes

Breast cancer (BC) continues to be the most frequently diagnosed cancer in American women, which disproportionately affects women of African-American (AA) descent. Previously, we reported greater serum levels of resistin in AA BC patients relative to Caucasian-American (CA) patients, and established its role in growth and aggressiveness of breast tumor cells. Here we have investigated the role of resistin in BC-chemoresistance. MDA-MB-231 and MDA-MB-468 BC cells of CA and AA origin, respectively, were incubated with resistin prior to doxorubicin treatment. Our data suggest that resistin conferred chemoresistance to both BC cell lines; however, the effect on AA cells was more profound. Furthermore, the resistin-induced doxorubicin-resistance was shown to occur due to suppression of apoptosis. Resistin treatment also affected the stemness of BC cells, as suggested by reduced cell surface expression of CD24, induced expression of CD44 and ALDH1, and increased capability of cells to form mammospheres. Mechanistic studies revealed that resistin-induced chemoresistance, apoptosis and stemness of BC cells were mediated through STAT3 activation. Taken together, our findings provide novel insight into the role of resistin in BC biology, and strengthen its role in racially disparate clinical outcomes.

ETV4 Facilitates Cell Cycle Progression in Pancreatic Cells through Transcriptional Regulation of Cyclin D1

The ETS family transcription factor ETV4 is aberrantly expressed in a variety of human tumors and plays an important role in carcinogenesis through upregulation of relevant target gene expression. Here, it is demonstrated that ETV4 is overexpressed in pancreatic cancer tissues as compared with the normal pancreas, and is associated with enhanced growth and rapid cell-cycle progression of pancreatic cancer cells. ETV4 expression was silenced through stable expression of a specific short hairpin RNA (shRNA) in two pancreatic cancer cell lines (ASPC1 and Colo357), while it was ectopically expressed in BXPC3 cells. Silencing of ETV4 in ASPC1 and Colo357 cells reduced the growth by 55.3% and 38.9%, respectively, while forced expression of ETV4 in BXPC3 cells increased the growth by 46.8% in comparison with respective control cells. Furthermore, ETV4-induced cell growth was facilitated by rapid transition of cells from G1- to S-phase of the cell cycle. Mechanistic studies revealed that ETV4 directly regulates the expression of Cyclin D1 CCND1, a protein crucial for cell-cycle progression from G1- to S-phase. These effects on the growth and cell cycle were reversed by the forced expression of Cyclin D1 in ETV4-silenced pancreatic cancer cells. Altogether, these data provide the first experimental evidence for a functional role of ETV4 in pancreatic cancer growth and cell-cycle progression. Implications: The functional and mechanistic data presented here regarding ETV4 in pancreatic cancer growth and cell-cycle progression suggest that ETV4 could serve as a potential biomarker and novel target for pancreatic cancer therapy. Mol Cancer Res; 16(2); 187–96. ©2017 AACR.

Gemcitabine treatment promotes immunosuppressive microenvironment in pancreatic tumors by supporting the infiltration, growth, and polarization of macrophages

Chemotherapy-induced immunosuppression poses an additional challenge to its limited efficacy in pancreatic cancer (PC). Here we investigated the effect of gemcitabine on macrophages, which are the first line of immune-defense mechanisms. We observed an increased presence of macrophages in orthotopic human pancreatic tumor xenografts from mice treated with gemcitabine as compared to those from vehicle only-treated mice. Conditioned media from gemcitabine-treated PC cells (Gem-CM) promoted growth, migration and invasion of RAW264.7 macrophage. In addition, Gem-CM also induced upregulation of M2-polarized macrophage markers, arginase-1 and TGF-β1. Cytokine profiling of gemcitabine-treated PC cells identified IL-8 as the most differentially-expressed cytokine. Incubation of Gem-CM with IL-8 neutralizing antibody diminished its ability to induce growth, migration and invasion of RAW264.7 macrophages, but did not abrogate their M2 polarization. Together, our findings identify IL-8 as an important mediator in the gemcitabine-induced infiltration of macrophages within the pancreatic tumor microenvironment and suggest the requirement of additional mechanism(s) for macrophage polarization.