Investigation of BCL-2 interacting killer (BIK) as a breast cancer biomarker and its role in failed apoptosis

Breast cancer is the number one cause of cancer-associated deaths in women worldwide. It can be broadly classified into estrogen receptor (ER) positive or -negative subtypes which form the basis of treatment. Despite the therapeutic advancements, more than half a million women annually succumb to the illness suggesting critical gaps in our understanding of the disease. Chemo-, radiation- and hormonal therapies are used to reduce tumor burden work by inducing apoptosis. Hence investigation of players in the apoptotic program can uncover the mechanisms of cancer aggression. Apoptosis is regulated by the BCL-2 family of proteins, which sense and relay the death signals to the downstream machinery that executes cell death. The BH3-only proteins, a subgroup of the BCL-2 family, act as the sensors of a variety of cell-intrinsic stressors and determine therapeutic response against cancer. Thus, it is believed that if the BH3-only proteins are absent or downregulated it may create a permissive environment for the development of therapeutic resistance leading to cancer aggression. In order to decipher how BH3 proteins affect breast cancer pathophysiology, I interrogated the gene expression levels of five BH3-only members in breast cancer tumors with respect to patient survival outcomes. Interestingly, we found that the BH3-only member BCL-2 interacting killer (BIK) was associated with worse patient survival and patients with high levels of BIK transcript were twice as likely to die from the disease compared to the BIK-low patients. This finding was surprising, given the pro-apoptotic nature of BIK. Hence, we validated these findings in a different cohort of patients by quantitating BIK protein levels using immunohistochemistry. Strikingly, BIK protein levels were also associated with worse survival outcomes of the patients. Thus, high BIK expression in two independent patient cohorts (total n=327) was associated with poor patient survival. Further interrogation revealed that high BIK mRNA levels in association with high levels of ATG5, a regulator of the cytoprotective autophagic pathway, predicted worse prognostic outcomes. This provided a clue that BIK might have a tumor-promoting effect through enhanced cell survival in the face of tumor-associated stress. In order to test this, I ectopically expressed BIK in breast cancer cells and tested their autophagic response to nutrient starvation or mammalian target of rapamycin complex 1 (mTORC1) inhibition. While there was some autophagic response, BIK did not stimulate robust autophagy. Furthermore, MTT assays revealed that BIK expression did not confer a growth or survival benefit on the cells when they were nutrient starved or rapamycin-treated. Together these observations showed that BIK did not strongly regulate autophagy in the cell lines tested yet hinted that BIK may have tumor-promoting effects in patients. More than 70% of all breast cancers are ER-positive driven by the female sex hormone estrogen. This type of cancer is treated with anti-estrogen therapy that utilizes drugs such as tamoxifen to induce cancer cell apoptosis and prevent relapse. Recent studies have identified an aberration of apoptosis called “failed apoptosis” in which apoptosis is initiated but does not go to completion, causes DNA double-strand breaks and mutations leading to tumorigenesis. Importantly, BIK is upregulated in response to anti-estrogen treatment of the ER-positive breast cancer which prompted me to interrogate BIK’s involvement in this patient subgroup. Interestingly, we discovered that high BIK levels predicted poor prognosis of anti-estrogen treated ER-positive but not alternatively treated ER-negative patients. I hypothesized that BIK facilitated tumor aggression in the ER-positive patients through failed apoptosis. Indeed, BIK expression in breast cancer cell lines led to minimal cell death but caused caspase activation, and double-strand DNA damage dependent on caspase activated DNAse (CAD). Importantly, tamoxifen-mediated BIK expression also induced genomic damage in cells, suggesting a link between anti-estrogen stimulated DNA damage-mediated mutagenesis. Finally, I explored whether transient expression of BIK altered cell phenotypes after long-term culture. Interestingly, cells surviving this treatment produced progeny with aggressive phenotypes characterized by high clonogenic survival, elevated anchorage-independent growth, and enrichment of cancer stem cells. These results suggest that poor survivals of BIK-high ER-positive patients could be partly due to failed apoptosis which may provoke mutations and provide a means of evolution for cancer cells. Thus, in this thesis, I identified BIK as a novel biomarker of breast cancer and discovered a potential mechanism of BIK-mediated tumor evolution

Connected by the cord—comparison of drug positivity rates and concentrations in a large cohort of paired umbilical cord and meconium specimens

Abstract Substance abuse during pregnancy has significant short- and long-term health, social, and legal implications. Hence, prompt determination of a neonate’s drug exposure status can aid the healthcare team in making treatment choices. Umbilical cord tissue (UC) and meconium are two commonly used specimens to detect fetal drug exposure. However, the current evidence on the sensitivity of one specimen over the other is both limited and conflicting. By utilizing a large cohort (n=4036) of paired UC and meconium samples originating from 13 states, we retrospectively investigated the qualitative and quantitative drug positivity rates for 31 analytes from 5 drug classes (i.e. cannabis, opioids, stimulants, barbiturates, and benzodiazepines). Semi-quantitative drug detection in UC samples was conducted by LC-MS-based assays, whereas an immunoassay screen preceded quantitative LC-MS analysis for meconium specimens. Data were analyzed using R-programming, Microsoft Excel, and GraphPad Prism packages. The overall drug positivity rate for UC was 56.57% compared to 52.38% in meconium. Opioids were the top drug class detected in UC at 35.53% compared to 20.64% in meconium. The detection rates for individual opioid analytes were also higher in UC (0.97-15.49%) relative to meconium (0.0 to 9.09%). Notably, 6-acetylmorphine (heroin metabolite) was only detected in UC (0.97%). In contrast, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC) was more frequently detected in meconium at 34.99% compared to 19.89% in UC. In the stimulant drug class, the overall amphetamine positivity rate was 7.73% in UC while 8.57% in the meconium specimens. Conversely, the cocaine positivity rate in the UC was 3.84% and 2.23% in meconium samples. In the barbiturate drug class, the positivity rate for butalbital was twice as high in UC (1.91%) relative to meconium (0.99%). Phenobarbital positivity rate was comparable in both matrices (~0.15%). The positivity rates for 6 common benzodiazepine drugs ranged from 0.40-0.94% in UC compared to 0.01-0.35% in meconium. Interestingly, a multidrug combination analysis revealed that THC−opioids was the most common two-drug combination detected in both matrices at a rate of 7.22% in UC compared to 5.39% in meconium. Subsequent examination of analyte concentrations between the two matrices revealed higher concentrations in meconium. THC was 48.13 times more concentrated in meconium relative to UC. Amphetamines and cocaine were 6.45-15.01 times more concentrated in meconium relative to UC. For the opioids, the overall concentrations were 21.61-1845.01 times higher in meconium specimens relative to UC. For barbiturates, butalbital had comparable concentrations in the two matrices whereas phenobarbital was 33.10 times more concentrated in the meconium. Lastly, benzodiazepines were overall 6.21-38.75 times more concentrated in meconium than UC. In conclusion, this study shows that despite most drugs being more concentrated in meconium, drug positivity rates were higher with UC for all analytes except THC and amphetamines, suggesting higher detection potential of the UC specimen.</jats:p

Can Umbilical Cord and Meconium Results Be Directly Compared? Analytical Approach Matters

Abstract Maternal drug use during pregnancy is a significant concern. Drug-exposed newborns are often born premature and may suffer from birth defects, neonatal abstinence syndrome and cognitive and developmental delays. Because of this, testing of neonatal specimens is carried out to assess fetal drug exposure during pregnancy. Umbilical cord tissue (UC) and meconium are commonly used specimens for this purpose. However, comprehensive studies comparing drug positivity rates and concentration in the two specimen types are lacking. To this end, 4,036 paired UC and meconium specimens originating from 13 states within the USA were identified, and retrospective analysis of drug positivity rates and drug concentration was performed for 31 analytes in 5 drug classes. Testing for 11-Nor-9-carboxy-tetrahydrocannabinol (THC-COOH) is a separate orderable for UC specimen at our laboratory, so a second data set was created for evaluation of this drug analyte with 2,112 paired UC and meconium specimens originating from 11 states. Testing of UC was performed by semi-quantitative liquid chromatography–tandem mass spectrometry (LC–MS-MS) assays, whereas, for meconium, an immunoassay-based screening preceded LC–MS-MS confirmation tests. Results generated for UC and meconium specimens were therefore compared for a total of 32 drug analytes from 6 drug classes. Drug concentrations for analytes were higher in meconium compared to UC, with the exception of phencyclidine. Despite this, the positivity rates for individual analytes were higher in UC, with the exception of THC-COOH and cocaine. Furthermore, analysis for multidrug positivity revealed that THC-COOH and opioids were the most common multidrug combination detected in both matrices. In conclusion, this study suggests that for most drug compounds, UC was more analytically sensitive to assess neonatal drug exposure by current methodologies. Additionally, by demonstrating that meconium has higher drug concentrations for most compounds, this study sets the stage for developing more sensitive assays in meconium.</jats:p

BIK drives an aggressive breast cancer phenotype through sublethal apoptosis and predicts poor prognosis of ER-positive breast cancer

AbstractApoptosis is fundamental to normal animal development and is the target for many anticancer therapies. Recent studies have explored the consequences of “failed apoptosis” where the apoptotic program is initiated but does not go to completion and does not cause cell death. Nevertheless, this failed apoptosis induces DNA double-strand breaks generating mutations that facilitate tumorigenesis. Whether failed apoptosis is relevant to clinical disease is unknown. BCL-2 interacting killer (BIK) is a stress-induced BH3-only protein that stimulates apoptosis in response to hormone and growth factor deprivation, hypoxia, and genomic stress. It was unclear whether BIK promotes or suppresses tumor survival within the context of breast cancer. We investigated this and show that BIK induces failed apoptosis with limited caspase activation and genomic damage in the absence of extensive cell death. Surviving cells acquire aggressive phenotypes characterized by enrichment of cancer stem-like cells, increased motility and increased clonogenic survival. Furthermore, by examining six independent cohorts of patients (total n = 969), we discovered that high BIK mRNA and protein levels predicted clinical relapse of Estrogen receptor (ER)-positive cancers, which account for almost 70% of all breast cancers diagnosed but had no predictive value for hormone receptor-negative (triple-negative) patients. Thus, this study identifies BIK as a biomarker for tumor recurrence of ER-positive patients and provides a potential mechanism whereby failed apoptosis contributes to cancer aggression.</jats:p