Firstly, we demonstrated that caspase-dependent cell death was enhanced by the over-expression of the type II TNF receptor (TNFR2).  HeLa cells, which naturally express high levels of type I TNF receptor (TNFR1) and low levels of TNFR2, were engineered to stably over-express TNFR2.  This combined with the use of genetically-engineered mutated TNFs that preferentially activate TNFR1 or TNFR2, we showed that both receptors can induce cell death, although this process occurred predominantly through TNFR1.  TNF-induced cell death was inhibited by the stable expression of cytokine response modifier A (CrmA), a potent inhibitor of receptor proximal caspases.  By isolating early apoptotic cells, we were able to identify differential activation profiles of members of the mitogen-activated protein kinase (MAPK) family during TNF-induced apoptosis.  In dying HeLa-TNFR2 cells, there was increased activation of c-Jun NH₂-terminal kinase (JNK) while the activation levels of p38 MAPK and p42/44 MAPK remained unchanged.  The use of peptidergic caspase inhibitors demonstrated that caspase-dependent modulation of JNK but not p38 MAPK or p42/44 MAPK takes place, and as such may provide a mechanism which accounts for the differences observed in MAPK activity during TNF-induced cell death.  Through expression of a dominant negative upstream activator of JNK (SEK-1-AL) and a pharmacological inhibitor of JNK activity, we were able to determine the role of JNK activation in TNF receptor-mediated apoptosis.  These findings clearly demonstrate that through cross-talk, TNF receptors, are able to further modulate the tightly regulate cellular consequences of TNF treatment and that JNK is a TNF-induced kinase that may play a role in the cytokine’s apoptotic cellular signalling.

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Lung cancer is the leading cause of cancer-related death in the United States and worldwide. Lung cancers that are driven by mutations in the Epidermal Growth Factor Receptor (EGFR) are treated with targeted anti-EGFR therapy, such as first-generation tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Unfortunately, all patients whose tumors initially respond to erlotinib or gefitinib will eventually go on to develop primary acquired resistance, most commonly through a second-site EGFR mutation, T790M. The combination of a second-generation EGFR TKI, afatinib, plus the anti-EGFR antibody, cetuximab, may overcome primary acquired resistance. Unfortunately, secondary acquired resistance is also seen with the new drug combination, and the underlying mechanisms have not yet been determined. Using tumor sample from a patient with secondary acquired resistance, we identified two unexpected NF2 mutations, neither of which was detected in the patients pre-treatment sample, suggesting that mutations in NF2 were acquired during treatment. The NF2 gene encodes merlin, a known tumor suppressor that is thought to regulate EGFR. Using cell line and animal models, we demonstrate that loss of NF2 is sufficient to induce resistance to TKI in EGFR-mutant cells. Co-treatment with the mTORC1 inhibitor, rapamycin, re-sensitizes cells to TKI. These studies suggest a novel potential mechanism of acquired resistance to targeted therapy in EGFR-mutant lung cancer. To further explore acquired resistance, we also evaluated a novel, irreversible, mutant-specific third-generation EGFR TKI, AZD9291. Pre-clinically, this drug potently inhibits signaling pathways and cellular growth in both EGFR-mutant and EGFR-mutant/T790M mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR mutant tumor xenograft and transgenic models. We use cell line and mouse models to characterize AZD9291, providing the foundation for a recently-initiated clinical trial. In sum, this work evaluates mechanisms of acquired resistance to targeted therapy in EGFR-mutant lung cancer and identifies potential strategies to overcome such resistance.

The purpose of the study was to learn more about the use or non use of the Papanicolaou Test (referred to hereafter as the Pap test) among a selected population of women on the Ball State University Campus.The author was concerned with the fact that, according to projections of the American Cancer Society, 12,000 women die each year of uterine cancer. Many of these deaths could have been avoided if women would have availed themselves yearly of a simple testing device known as the Pap test.After considerable research and discussions with Ball State University women of all ages, it was obvious that many women do not take advantage of this cancer detection device. It became this author's desire to discover why the Pap test is overlooked by so many women. Research pointed out several reasons for this phenomenon, namely: 1) length of time required for the test, 2) inability to afford the Pap test, 3) fear of the results and 4) lack of prior experience with the Pap test combined with a feeling of wellness. latter reason was summarized in the general statement: "Have never had the Pap test and I am fine, so why bother?".Also appearing in this research was the subtle hint that perhaps the medical profession itself was partly to blame for the nonuse of the test. Overworked physicians and understaffed laboratories were cited as possible reasons why doctors were not encouraging their patients to avail themselves of the Pap test.Previous studies on this subject have been conducted in lower socioeconomic populations in San Diego, California, and Baltimore, Maryland. The above named reasons for not having the yearly Pap test were the conclusions of these studies also. The author felt that a study of women representing a different segment of society possibly would be of help in this area of cancer research and control.A questionnaire was developed and distributed to the female residents in all the Ball State University married students' housing units. This selected group of women represented a fairly homogeneous population with an assumed similar type of background and education.A total of 670 questionnaires were distributed and 535 were completed and returned for an 80 per cent rate of response.Following are some of the significant results of the survey:83 per cent of the women had a complete physical examination within the last year.96 percent of the women had a Pap test within the last year.74 per cent of the women indicated their desire for a "clean bill of health" which prompted yearly Pap tests.89.9 per cent stated they were not afraid to learn the results of the Pap test.41 per cent of the women revealed that their first knowledge of the Pap test came through the news media.92 per cent of the women felt their husbands were more aware of the benefits of the Pap test than they themselves. The results of this study indicated that these women, as a group, were concerned about their health and were eager to seek out proper medical care in order to safeguard themselves.

There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-β) pathway-associated genes relative to other subtypes, including the TGF-β receptor type III (TβRIII). We hypothesized that TβRIII is tumor promoter in mesenchymal-stem like TNBC cells. Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of TβRIII in the MSL subtype. We stably expressed short hairpin RNAs specific to TβRIII (TβRIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine TβRIII expression patterns across all TNBC subtypes. TβRIII was the most differentially expressed TGF-β signaling gene in the MSL subtype. Silencing TβRIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when TβRIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed TβRIII-dependent phenotypes, we discovered that integrin-α2 was expressed at higher levels in MSL cells after TβRIII-KD. Stable knockdown of integrin-α2 in TβRIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells. We have found that TβRIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that TβRIII-KD elevates expression of integrin-α2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both TβRIII and integrin-α2. Overall, our results indicate a potential mechanism in which TβRIII modulates integrin-α2 expression to effect MSL cell migration, invasion, and tumorigenicity.

LIM domain Only-2 (LMO2) is a T cell oncogene whose molecular mechanism for T-cell transformation still remains to be elucidated. There are two hypotheses that could explain how the enforced expression of LMO2 induces T-cell Acute Lymphoblastic Leukemia (T-ALL). The first hypothesis is that LMO2 enforced expression results in the functional deficiency of E2A proteins. LMO2 overexpression correlates with the loss of E2A proteins, or Heb, suggesting that LMO2 functions by creating a deficiency of E2A either by redirection, sequestration, or turnover. To test this hypothesis, we enforced the expression ofE47Lmo2-induced T-ALL cell lines using an E47/estrogen receptor fusion construct that could be forced to homodimerize with 4-hydroxytamoxifen (4HT). We discovered that forced homodimerization triggered a G1 cell cycle growth arrest in 2 of the 4 lines tested. Transcriptome analysis suggested that E47 has remarkably diverse effects in T-ALL but that functional deficiency of E47 is not a universal feature of Lmo2-induced T-ALL. The second hypothesis, suggests that Lmo2 enforced expression results in transcriptional regulation of genes needed for T-cell transformation. The gene of interest in our studies is Hematopoietically expressed homeobox (Hhex). Hhex is a T-cell oncogene that is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. To further understand Hhexs function, we induced a conditional knockout in floxed Hhex mice with the Vav-iCre transgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhex in all hematopoietic tissues. Most impressively, Hhex conditional knockout markedly prolonged T-ALL onset in CD2-Lmo2 transgenic mice. Hhex conditional knockouts also had a significant decrease in mature B cells in the spleen and bone marrow. Bone marrow transplant and thymic repopulation studies revealed that loss of Hhex is important not only B cell development but also early T cell development. Our experiments show that Hhex is a critical transcription factor in lymphoid development and in LMO2-induced T-ALL.

Breast cancer is the leading cancer diagnosis in pre-menopausal women in the U.S. Of these breast cancers, 25% are diagnosed 2-5 years post-partum. Unfortunately, post-partum breast cancers (ppBCs) are highly metastatic. The reasons underlying the exaggerated lethality of ppBCs are unclear, but relate to stromal remodeling events that occur during post-partum involution, when milk-producing mammary epithelial cells (MECs) undergo widespread cell death. Upon engulfment of apoptotic cells (efferocytosis), immune suppressive wound healing cytokines are produced including Tgfβ1, IL4, and IL10, which increase macrophage polarization towards an M2 phenotype. Wound healing/TH2-like cytokines and M2 macrophages each correlate with decreased disease-free survival in breast cancer patients. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine that has gained interest for its role in both tumor progression and metastasis. TGFβ1 operates to suppress cytotoxic immunity, increase fibroblast activation and collagen deposition, increases migration and invasion of tumor epithelial cells, and may enhance cancer stem cell-like properties in some tumor cells. TGFβ1 is abundantly up-regulated in response to efferocytosis during post-partum involution of the mammary gland. We recently showed that TGFβ1 is induced upon macrophage-mediated efferocytosis in the mammary tumor microenvironment (TME). Secreted TGFβ1 also acts on mammary tumor epithelial cells to increase epithelial-mesenchymal transition (EMT), cell adhesion, motility, and invasion. We utilized an immune-competent mouse model of ppBC, which recapitulates many of the clinical aspects of ppBCs in patients, including increased metastasis in response to post-partum events to show that inhibition of efferocytosis for the first 7 days of involution prevents M2 macrophage polarization, blocks induction of TGFβ1, and prevents metastasis of ppBCs through involution day 40. Our preliminary results show that ppBCs treated with the neutralizing anti-TGFβ antibody 1D11 for the first 14 d of involution produce fewer lung metastases as compared to control IgG-treated ppBCs.

Cytogenetic analysis was performed on 40 non-Hodgkin's lymphoma (NHQ node biopsies. Chromosomes X, 3 and 12 were the most frequently gained; of the much rarer monosomies, loss of chromosome 13 was most common. Structural abnormalities primarily involved chromosomes 14,1,18,6 and 17. A markedly greater number of chromosome gains were associated with low-grade disease when compared to high-grade. In order to obtain further information from the cytogenetic analysis of the NHL karyotypes, the fluorescence in-situ hybridisation (FISH) technique was applied to the series. The activation state of additional X-chromosomes was examined and evidence that more than one X-chromosome was present in the active state in 4/9 cases was obtained. Further, in an apparent case of monosomy X, a marker was identified as an abnormal X-chromosome by chromosome painting. Interphase FISH was applied to NHL cells and numerical chromosome changes were identified; this approach was also attempted on aged bone marrow smears from acute lymphocytic leukaemia patients, in order to test the utility of the technique on archival material. Dual chromosome painting was used to elucidate the origins of add(14) chromosomes in 8 of the cases. In the control and two other cases the translocated material was demonstrated to be from chromosome 18, in two cases it was from chromosome 3 and in one case them was an insertion of chromosome 11 material. it was not possible to identify the origins of the translocated material in one NHL and in the final case the apparent add(14) was demonstrated not to contain chromosome 14 material. Structural abnormalities of chromosome 6 were investigated both by chromosome painting and by hybridisation of the MYB gene. The latter, which was initially mapped to 6q23 before hybridisation to NHL cells revealed previously unsuspected rearrangements. One case contained extrachromosomal chromatin bodies that appeared to be double minute chromosomes (dmin), which FISH analysis demonstrated to be derived from the X-chromosome and contain centromere-associated DNA. The significance of these results is discussed with reference to previously published series of NHL karyotypes.

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