Browsing by Subject "Tumor recurrence"

Whole-genome duplication (WGD) generates polyploid cells possessing more than two copies of the genome. These events commonly occur during the evolution of human tumors across tissue types and mutational drivers, affecting an estimated 30-37% of all tumors. The frequency of WGD increases in advanced and metastatic tumors, and WGD is associated with poor prognosis in diverse tumor types, suggesting a functional role for polyploidy in tumor progression. Experimental evidence suggests that polyploidy has both tumor-promoting and suppressing effects. The polyploidization of a normally diploid cells often compromises genomic stability. In this way, WGD may be capable of promoting tumor formation, growth and progression, by facilitating the evolution of genetic heterogeneity on which selection can act. However, while some features of polyploidy can promote tumor growth, these features can also be countered by associated tumor suppressive qualities of polyploidization and associated cellular stresses. Chromosomal instability and resulting aneuploidy often have negative effects on cellular fitness; this can occur through the induction of proteotoxic stress, replication stress and delayed proliferation. Polyploidization can also be opposed by cell intrinsic and extrinsic pathways, including p53, the Hippo pathway and immunosurveillance. How these diverse and multifaceted features of polyploid cells work together to regulate tumor progression remains unclear.

Using a genetically engineered mouse model of HER2-driven breast cancer, we explored the prevalence and consequences of whole-genome duplication during tumor growth and recurrence. While primary tumors in this model are invariably diploid, nearly 40% of recurrent tumors undergo WGD. WGD in recurrent tumors was associated with increased chromosomal instability, decreased rates of proliferation and increased survival in stress conditions. The effects of WGD on tumor growth were dependent on tumor stage. Surprisingly, in recurrent tumor cells, WGD slowed tumor formation, tumor growth rate and opposed the process of recurrence, while WGD promoted the growth of primary tumors. Our findings highlight the importance of identifying conditions that promote the growth of polyploid tumors, including the cooperating genetic mutations that allow cells to overcome the barriers to WGD tumor cell growth and proliferation.

While our results revealed fitness disadvantages for recurrent polyploid tumor cells, the paradox remains that WGD is common in cancer cells despite this, suggesting that cells must evolve ways to overcome barriers to tumorigenesis. These findings suggest that a polyploid cancer cell may be delicately balanced, relying on certain pathways or processes to compensate for its cellular deficiencies more than their diploid counterparts. Ploidy-specific lethality describes the phenomenon in which inhibiting the activity or expression of a specific protein results in death of polyploid cells but not their diploid counterparts. To interrogate this idea, we next employed our models of recurrent polyploid cells to explore the impact of polyploidization on gene expression and signaling dependencies. Using RNA sequencing we uncovered that tetraploid cells exhibited decreased expression of genes of the cGAS-STING pathway. We performed two loss-of-function CRISPR screens against the kinome, one in vitro and one in vivo, to identify ploidy-specific lethal genes. The in vivo screen revealed candidates for ploidy-specific lethal genes including Srpk1, Mark4 and Ryk. Together these results demonstrated that polyploid recurrent tumor cells exhibit unique gene expression patterns that may reflect selection pressure of the immune system and may rely on unique survival mechanisms in vivo.

The survival and eventual recurrence of dormant residual tumor cells following therapy is a leading cause of death in many tumor types. The metabolic properties of dormant residual tumor cells, which are thought to be quiescent or slowly proliferating, are likely distinct from those of rapidly growing tumors. However, it is not known whether alterations in cellular metabolism directly regulate the survival of dormant cells or their reactivation to form recurrent tumors. To address this, we used a conditional mouse model of Her2-driven breast cancer to study metabolic adaptations following Her2 inhibition, during dormancy, and after tumor recurrence. First, we found that Her2 downregulation caused widespread changes in cellular metabolism, culminating in oxidative stress. Tumor cells adapted to this metabolic stress by upregulation of the antioxidant transcription factor, NRF2. Constitutive NRF2 expression persisted during dormancy and in recurrent tumors. Constitutive activation of NRF2 accelerated recurrence, while suppression of NRF2 impaired recurrent tumor growth. These results are supported by clinical data showing that the NRF2 transcriptional program is activated in recurrent breast tumors, and that NRF2 is associated with poor prognosis in patients with breast cancer. Mechanistically, NRF2 signaling in recurrent tumors induced metabolic reprogramming to re-establish redox homeostasis and upregulate de novo nucleotide synthesis. An in vivo CRISPR screen identified genes in the redox and nucleotide pathways as the essential downstream mediators of NRF2 in recurrent tumors. The NRF2-driven metabolic state rendered recurrent tumor cells sensitive to glutaminase inhibition, and glutaminase inhibition prevented reactivation of dormant tumor cells, suggesting that NRF2-high dormant and recurrent tumors can be therapeutically targeted. Together, these data provide evidence that NRF2-driven metabolic reprogramming promotes the recurrence of dormant breast cancer. Second, we found that the metabolic enzyme Bcat1 is upregulated in recurrent tumors as a result of epithelial-to-mesenchymal transition. We found that Bcat1 knockout impaired recurrent tumor growth, demonstrating its potential as a therapeutic target for recurrent tumors.

Advances in anti-cancer therapies, including chemotherapies, targeted therapies, and immuno-therapies, have drastically improved patient outcomes over the last few decades. However, tumor recurrence and acquired drug resistance continue to be detrimental for cancer patients, accounting for the majority of cancer-related deaths. Drug resistance is a common phenomenon that occurs when pharmaceutical agents are tolerated by and no longer effective against their target. In the case of cancer, acquired drug resistance is when cancer cells are able to survive, actively proliferate, and migrate to distant sites in the presence of anti-cancer agents. As such, it is imperative that we understand the mechanisms that lead to acquired drug resistance and tumor recurrence and leverage this newfound understanding to improve treatment options and, ultimately, patient outcomes.

Recurrent tumors and drug resistant cancer cells arise from drug-tolerant persister cells (DTPs), a population of cells that is able to withstand and adapt to cancer treatment. APOBEC3 and NRF2 are proteins that are largely absent and inactive in primary non-small cell lung cancer and HER2+ breast cancer, respectively. Interestingly, both proteins are found in recurrent disease, specifically after targeted therapy. This suggests that the upregulation of APOBEC3 and NRF2 is an adaptive response to anti-cancer treatment. Further, this suggests that APOBEC3 and NRF2 are essential for drug-tolerant persister cell survival and subsequent recurrence. Understanding how these proteins are induced, regulated, and what their respective roles are in tumor evolution can provide insight into therapeutic potentials in both residual and recurrent disease. First, I explored the regulation and function of APOBEC mutagenesis during acquired resistance to epidermal growth factor receptor (EGFR) inhibitors in two non-small cell lung cancer cell lines, PC9 and HCC827. I assess the effects of EGFR inhibition on APOBEC3 expression and activity and find that this induces APOBEC3 activity. Moreover, I evaluate how sustained APOBEC3 activity promotes evolution of drug resistance in DTPs. Finally, I examine what mechanisms might play a role in acquired drug resistance when APOBEC3 is highly active. I show that while APOBEC activity does not accelerate acquired therapy resistance, A3B expression alters the evolutionary path that PC9 cells take to become gefitinib-resistant. Specifically, A3B expression is associated with the late acquisition of T790M mutations during the DTP state, supporting a model where induction of APOBEC activity promotes DTP survival, thereby facilitating the on-going evolution of drug-tolerant persister cells. Of note, I find that APOBEC3 activity is associated with squamous cell transdifferentiation in PC9 cells, suggesting that p63 and its target genes could be future biomarkers and therapeutic targets.

Second, I investigated the regulation of the transcription factor NRF2 in recurrent breast cancer cells. I assess KEAP1-mediated regulation of NRF2 and find that while KEAP1 knockout in primary and recurrent cells caused an increase in NRF2 and its target genes, NRF2 remained more elevated in recurrent cells, indicating that increased NRF2 levels and transcriptional activity in these cells are independent of KEAP1. Next, I looked at post-translational modifications on NRF2 to determine if this may be the cause of the differential NRF2 levels in primary and recurrent cells. I found that NRF2 in recurrent cells had higher levels of phospho-Ser364, potentially affecting NRF2’s stability. Finally, I evaluated regulation of NRF2 by Akt and GSK-3β. I found that inhibition of Akt had no effect on NRF2 levels. In contrast to this, I found that GSK-3β activity is inversely correlated with NRF2 levels, suggesting that low levels of GSK-3β activity is partially responsible for NRF2 stabilization in recurrent tumor cells.

In conclusion, I have modeled two adaptive mechanisms for tumor recurrence and acquired drug resistance in two different cancer types. I elucidated the mechanisms by which APOBEC3B and NRF2 are able to promote cancer cell evolution in drug-tolerant persister cells that eventually give rise to recurrences.