Targeting the Microenvironment: Approaches for Reducing Breast Cancer Metastases and Chemotherapy-induced Bone Loss

The tumor microenvironment plays a pivotal role in tumor progression, and there is mounting evidence for synergistic action between tumor cells and microenvironmental cells that help the former thrive and even metastasize to other organs. This dissertation is focused on investigating the potential of therapeutically targeting the microenvironment to reduce breast cancer metastases and chemotherapy-induced bone loss. In Chapter 2 we investigate the efficacy of p38MAPK/MK2 inhibitors in limiting breast cancer metastasis by targeting the stromal compartment. Chapter 3 is centered around identification of mechanisms that drive chemotherapy-induced bone loss and the potential role for chemotherapy-induced senescence in this bone loss. Tumor-associated stromal cells are notorious for secreting factors that have pro-tumorigenic capabilities. The stress kinase, p38MAPK, is known to regulate the expression of several of these tumor-promoting stromal factors that in fact, have been found in the stroma of breast cancer patients. Moreover, p38MAPK_ is frequently upregulated in a variety of cancers including breast cancer, and its expression correlates with poor prognosis. In earlier work, we demonstrated that inhibition of p38MAPK resulted in attenuated primary tumor growth and this effect is through blocking secretion of stromal factors. This effect at the primary site, led us to investigate whether inhibition of p38MAPK_ could reduce breast cancer metastases in a clinically relevant model. In this dissertation, we show that orally administered, small-molecule inhibitors of p38MAPK_ and its downstream kinase, MK2, each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. We demonstrate that this effect is due to the inhibition of the p38MAPK_ pathway within the stromal compartment. In addition, these inhibitors effectively inhibited tumor-associated and chemotherapy-induced bone loss, which represent significant comorbidities for cancer patients. We show that targeting the stromal p38MAPK-MK2 pathway limits metastatic breast cancer growth while preserving bone quality and extending survival. The second part of this dissertation focuses on investigating estrogen-independent mechanisms that drive bone loss following chemotherapy treatment. Patients who receive chemotherapy treatment experience significant loss in bone density. Chemotherapy is thought to lead to bone loss by inducing premature menopause, which subsequently results in estrogen loss. Given that estrogen plays an important role in bone homeostasis, the loss of ovarian function is thought to drive bone loss in women undergoing chemotherapy. While it is clear estrogen is important in bone homeostasis and its loss following chemotherapy contributes to bone loss, these observations do not fully explain why post-menopausal women, who have already undergone physiological ovarian failure, lose significant bone density following therapy. Further, therapy-induced bone loss is more rapid and severe than bone loss seen with aging or post-menopause. Together, these observations suggest that factors or mechanisms in addition to estrogen loss contribute to chemotherapy-induced bone loss. Given that chemotherapeutic agents robustly induce senescence in vitro and in vivo, we postulated that chemotherapy-induced senescence and SASP activation contributes to bone loss. We used a Doxorubicin-induced bone loss model in mice and observed that chemotherapy reduced bone volume significantly more than the classic OVX model of estrogen deficiency both in young (6-week old) and skeletally mature (16-week old) mice in C57BL/6 and FVB/NJ strains. To evaluate the role of senescence in chemotherapy-induced bone loss, we profiled the expression of several senescence-associated factors in bones devoid of marrow. We observed induction of senescence marker p16, and factors including IL-6 and Dkk1, in 6-week old mice at the early (48 hour post Doxorubicin) timepoint and 16-week old chemotherapy mice at the final (10 days post Doxorubicin) timepoint. However, the upregulation did not repeat in 16-week old mice, leaving some questions of inconsistencies that will be addressed in prospective studies. In line with our hypothesis, we treated mice with p38MAPK and MK2 inhibitors to block production of senescence-associated proresorptive factors and observed preservation of bone integrity in chemotherapy-treated mice. In conclusion, we established that there are estrogen-independent mechanisms involved in chemotherapy-induced bone loss. However, the contribution of senescence and the mechanism of bone preservation by p38i and MK2Pi requires further evaluation. Overall, targeting the microenvironment with the intent of disrupting the stromal-tumoral collaboration may be a viable therapeutic strategy to combat tumor progression and related pathologies that result from cancer treatments

Low Phosphate Alters Lateral Root Setpoint Angle and Gravitropism

Premise of the study: Lateral roots, responsible for water and nutrient uptake, maintain nonvertical angles throughout development. Soil phosphate is one limiting nutrient for plant growth that is known to induce changes to root system architecture, such as increased lateral root formation. This study seeks to determine whether phosphate concentration affects lateral root orientation in addition to its previously described influences on root architecture. Methods: Images of intact Arabidopsis root systems were recorded for 24 h, and lateral root tip angles were measured for wild‐type and mutant pgm‐1 and pin3‐1 roots on a full or low phosphate medium. Setpoint angles of unstimulated root systems were determined, as were gravitropic responses of lateral roots over time. Key results: The root system setpoint angles of wild‐type and mutant pin3‐1 roots showed a shift toward a more vertical orientation on low orthophosphate (Pi) medium. The gravitropic responses of both pgm‐1 and pin3‐1 roots on low Pi medium was elevated relative to control Pi medium. Mutations in two phosphate transporters with high levels of expression in the root showed a gravitropic response similar to wild‐type roots grown on low Pi, supporting a role for Pi status in regulating lateral root gravitropism. Conclusions: Lateral root orientation and gravitropism are affected by Pi status and may provide an important additional parameter for describing root responses to low Pi. The data also support the conclusion that gravitropic setpoint angle reacts to nutrient status and is under dynamic regulation

Identification and prioritisation of potential vaccine candidates using subtractive proteomics and designing of a multi-epitope vaccine against Wuchereria bancrofti

Abstract This study employed subtractive proteomics and immunoinformatics to analyze the Wuchereria bancrofti proteome and identify potential therapeutic targets, with a focus on designing a vaccine against the parasite species. A comprehensive bioinformatics analysis of the parasite's proteome identified 51 probable therapeutic targets, among which "Kunitz/bovine pancreatic trypsin inhibitor domain-containing protein" was identified as the most promising vaccine candidate. The candidate protein was used to design a multi-epitope vaccine, incorporating B-cell and T-cell epitopes identified through various tools. The vaccine construct underwent extensive analysis of its antigenic, physical, and chemical features, including the determination of secondary and tertiary structures. Docking and molecular dynamics simulations were performed with HLA alleles, Toll-like receptor 4 (TLR4), and TLR3 to assess its potential to elicit the human immune response. Immune simulation analysis confirmed the predicted vaccine’s strong binding affinity with immunoglobulins, indicating its potential efficacy in generating an immune response. However, experimental validation and testing of this multi-epitope vaccine construct would be needed to assess its potential against W. bancrofti and even for a broader range of lymphatic filarial infections given the similarities between W. bancrofti and Brugia

Parasellar Chondroid Chordoma: a Case Report

Chordomas are distinct tumors which arise almost exclusively in the midline skeleton, from skull base to the sacrum. Chondroid chordoma is a rare form of chordoma with features of chordoma and chondrosarcoma arising almost at skull base. We report a case of a right parasellar chondroid chordoma in 54 years old female. Histopathology and IHC confirm the diagnosis of this case

Stromal-Initiated Changes in the Bone Promote Metastatic Niche Development

More than 85% of advanced breast cancer patients suffer from metastatic bone lesions, yet the mechanisms that facilitate these metastases remain poorly understood. Recent studies suggest that tumor-derived factors initiate changes within the tumor microenvironment to facilitate metastasis. However, whether stromal-initiated changes are sufficient to drive increased metastasis in the bone remains an open question. Thus, we developed a model to induce reactive senescent osteoblasts and found that they increased breast cancer colonization of the bone. Analysis of senescent osteoblasts revealed that they failed to mineralize bone matrix and increased local osteoclastogenesis, the latter process being driven by the senescence-associated secretory phenotype factor, IL-6. Neutralization of IL-6 was sufficient to limit senescence-induced osteoclastogenesis and tumor cell localization to bone, thereby reducing tumor burden. Together, these data suggest that a reactive stromal compartment can condition the niche, in the absence of tumor-derived signals, to facilitate metastatic tumor growth in the bone