Geometric tumor embolic budding characterizes inflammatory breast cancer.

PURPOSE: Inflammatory breast cancer (IBC) is characterized by numerous tumor emboli especially within dermal lymphatics. The explanation remains a mystery. METHODS: This study combines experimental studies with two different IBC xenografts with image algorithmic studies utilizing human tissue microarrays (TMAs) of IBC vs non-IBC cases to support a novel hypothesis to explain IBC's sina qua non signature of florid lymphovascular emboli. RESULTS: In the human TMAs, compared to tumor features like nuclear grade (size), mitosis and Ki-67 immunoreactivity which show that IBC is only modestly more proliferative with larger nuclei than non-IBC, what really sets IBC apart is the markedly greater number of tumor emboli and distinctly smaller emboli whose numbers indicate geometric or exponential differences between IBC and non-IBC. In the experimental xenograft studies, Mary-X gives rise to tight spheroids in vitro which exhibit dynamic budding into smaller daughter spheroids whereas Karen-X exhibits only loose non-budding aggregates. Furthermore Mary-X emboli also bud dramatically into smaller daughter emboli in vivo. The mechanism that regulates this involves the generation of E-cad/NTF1, a calpain-mediated cleavage 100 kDa product of 120 kDa full length membrane E-cadherin. Inhibiting this calpain-mediated cleavage of E-cadherin by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides that both penetrate the cell membrane and mimic either the cleavage site or the binding site on E-cadherin, inhibits the generation of E-cad/NTF1 in a dose-dependent manner, reduces spheroid compactness and decreases budding. CONCLUSION: Since E-cad/NFT1 retains the p120ctn binding site but loses the α-and ß-catenin sites, promoting its 360° distribution around the cell's membrane, the vacilating levels of this molecule trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of emboli seen in IBC.

A preliminary study of the probitive value of personality assessment in medical school admissions within the United States.

BACKGROUND: Allopathic medicine faces a daunting challenge of selecting the best applicants because of the very high applicant / matriculant ratio. The quality of graduates ultimately reflects the quality of medical practice. Alarming recent trends in physician burnout, misconduct and suicide raise questions of whether we are selecting the right candidates. The United States (US) lags far behind the United Kingdom (UK) and Europe in the study of non-cognitive tests in medical school admissions. Although more recently, medical schools in both the UK, Europe and the US have begun to use situational judgement tests such as the Computer-Based Assessment for Sampling Personal Characteristics (CASPer) and the situational judgement test (SJT), recently developed by the Association of American Medical Colleges (AAMC) and that these tests are, in a sense non-cognitive in nature, direct personality tests per se have not been utilized. We have historically used, in the admissions process within the US, knowledge, reasoning and exam performance, all of which are largely influenced by intelligence and also improved with practice. Personality, though also undoubtedly influenced by intelligence, is fundamentally different and subject to different kinds of measurements. METHODS: A popular personality measurement used over the past two decades within the US in business and industry, but not medical school has been the Neo Personality Inventory - Revised (NEO-PI-R) Test. This test has not been utilized regularly in allopathic medicine probably because of the paucity of exploratory retrospective and validating prospective studies. The hypothesis which we tested was whether NEO-PI-R traits exhibited consistency between two institutions and whether their measurements showed probative value in predicting academic performance. RESULTS: Our retrospective findings indicated both interinstitutional consistencies and both positive and negative predictive values for certain traits whose correlative strengths exceeded traditional premed metrics: medical college admission test (MCAT) scores, grade point average (GPA), etc. for early academic performance. CONCLUSIONS: Our exploratory studies should catalyze larger and more detailed confirmatory studies designed to validate the importance of personality traits not only in predicting early medical school performance but also later performance in one's overall medical career.

The virtues of the virtual medical school interview.

The COVID-19 pandemic has mandated the use of virtual interactions in medical school. Although this falls mainly in the area of didactic instruction, of necessity, it has extended to the critical Admissions Process and the Medical School Interview itself. The California University of Science and Medicine (CUSM) with their flipped classroom approach had previously entered a virtual space of instruction even before COVID-19. Because CUSM was, in a sense, already committed to 'virtual' education, in the face of the COVID-19 pandemic, CUSM focused not on what it might lose but what it might gain and what their applicants to medical school might gain with the virtual format. The COVID-19 pandemic provided a unique opportunity to initially compare the Virtual Interview with the traditional On-Campus (In-Person) Interview during the hybrid 2020 year when the COVID-19 pandemic began. The Virtual Interview was patterned after the On-Campus Interview with some modifications. The same faculty conducted both interviews. A number of inherent advantages of the Virtual Interview surfaced to these faculty interviewers based on their subjective observations and conclusions. The overall interviewee satisfaction with the Virtual Interview was very positive based on their subjective observations and conclusions. The objective data from the Virtual Interviews compared to the On-Campus Interviews in the hybrid year resulted in a greater percentage of both offers of acceptance (p = .001) and matriculations (p = .001). In order to strengthen our initial observations, we expanded our study to include 2 pre-COVID-19 years (2018, 2019) of exclusively On-Campus interviews (n = 743) and 1 additional COVID-19 year (2021) of exclusively Virtual Interviews (n = 529). In this expanded study, interviewee demographics were not confounding and the Virtual Interview gave rise to overall greater interviewee satisfaction (p = .001), a trend to greater interviewer satisfaction and a greater percentage of both offers of acceptance (p = .047) and matriculations (p = .036).

The cell of cancer origin provides the most reliable roadmap to its diagnosis, prognosis (biology) and therapy.

Cancers arise from single transformed cells from virtually every organ of the body, divide in a relatively uncontrolled manner, and metastasize widely. A search for a "magic bullet" to precisely diagnose, characterize, and ultimately treat cancer has largely failed because cancer cells do not differ significantly from their organ-specific cells of origin. Instead of searching for genomic, epigenetic, transcriptional, and translational differences between cancers and their cells of origin, we should paradoxically focus on what cancer cells have in common with their untransformed cells of origin. This redirected search will lead to improved diagnostic and therapeutic strategies where therapeutic index considerations and drug-limiting toxicities can largely be circumvented. We cite three cancer examples that illustrate this paradigm-shifting strategy: pseudomyxoma peritonei (PP), metastasis of unknown origin (cancers of unknown primary) (MUO), and cancers that arise from potentially dispensable organs (CAD). In each of these examples, the cell of cancer origin still provides the most reliable road map to its diagnosis, prognosis (biology), and therapy.

Use of constitutive and inducible oncogene-containing iPSCs as surrogates for transgenic mice to study breast oncogenesis.

BACKGROUND: Powerful constitutive and inducible transgenic / bitransgenic / tritransgenic murine models of breast cancer have been used over the past two decades to shed light on the molecular mechanisms by which the given transgenic oncogenes have interacted with other cellular genes and set in motion breast cancer initiation and progression. However, these transgenic models, as in vivo models only, are expensive and restrictive in the opportunities they provide to manipulate the experimental variables that would enable a better understanding of the molecular events related to initial transformation and the target cell being transformed. METHODS: To overcome some of these limitations, we derived oncogene-containing induced pluripotent stem cell (iPSC) clones from tail vein fibroblasts of these transgenic mice and manipulated them both in vitro and in vivo in non-transgenic background mice. We created the iPSC clones with a relatively low M.O.I, producing retroviral integrations which averaged only 1 to 2 sites per retroviral plasmid construct used. RESULTS: Most iPSC clones derived from each group displayed an essentially normal murine karyotype, strong expression of the exogenous reprogrammable genes and significant expression of characteristic endogenous murine surface stem cell markers including SSEA-1 (CD15), PECAM-1 (CD31), Ep-Cam (CD326), and Nectin (CD112), but no expression of their transgene. A majority (75%) of iPSC clones displayed a normal murine karyotype but 25% exhibited a genomically unstable karyotype. However, even these later clones exhibited stable and characteristic iPSC properties. When injected orthotopically, select iPSC clones, either constitutive or inducible, no longer expressed their exogenous pluripotency reprogramming factors but expressed their oncogenic transgene (PyVT or ErbB2) and participated in both breast ontogenesis and subsequent oncogenesis. When injected non-orthotopically or when differentiated in vitro along several different non-mammary lineages of differentiation, the iPSC clones failed to do so. Although many clones developed anticipated teratomas, select iPSC clones under the appropriate constitutive or inducible conditions exhibited both breast ontogenesis and oncogenesis through the same stages as exhibited by their transgenic murine parents and, as such, represent transgenic surrogates. CONCLUSIONS: The iPSC clones offer a number of advantages over transgenic mice including cost, the ability to manipulate and tag in vitro, and create an in vitro model of breast ontogeny and oncogenesis that can be used to gain additional insights into the differentiated status of the target cell which is susceptible to transformation. In addition, the use of these oncogene-containing iPSC clones can be used in chemopreventive studies of breast cancer.

Why is cancer so common a disease in people yet so rare at a cellular level?

Cancers are common diseases in people and yet, on a cellular level, are quite rare. The vast majority of both sporadic, spontaneous cancers and inherited germline cancers arise in single foci from singly transformed cells despite the fact that, in the former, carcinogenic factors bathe fields of millions of potential target cells and, in the latter, the predisposing germline mutations are present in every cell of a given organ and, in fact, every cell of the body. Although the multi-hit theory of carcinogenesis has been invoked to explain such things as cancer latency, which is the period between cancer initiation and emergence and the cancer-aging relationship where an accumulation of "hits" over a period of time are necessary for cancer emergence, the multi-hit theory falls short in explaining the rareness of transformation at a cellular level. This is so because many cancers are not due to multiple hits, and even for those that are, it would be expected that many cells would be exposed to those factors inducing the hits. Although the tumor stem/progenitor cell compartmental theory of tumorigenesis characterizes a tumor compartment that is capable of self-renewal and multipotency, accounting for cancer relapses and recurrences, this compartmental theory alone cannot account for the rareness of initial transformation at a cellular level as the cancer stem/progenitor cell compartment is already transformed and considerable in size. This study advances a different and novel hypothesis that oncogenesis is regulated and ultimately determined by a cell of origin's critical state of differentiation. Before and after this critical state of differentiation has been reached, target cells cannot transform and give rise to cancer even when they receive the necessary carcinogenic insults or have the requisite transforming tumor suppressor genes or oncogenes. As support for this hypothesis, the study cites preliminary evidence using oncogene-containing transgenic mice that develop mammary carcinomas, to derive tail vein fibroblasts converted to iPSCs which, when left undifferentiated, and injected into the cleared fat pads of non-transgenic background mice give rise to mammary gland ontogeny and mammary gland carcinogenesis. However, when first differentiated in vitro into multiply different non-mammary lineages prior to injection, they fail to do so. The hypothesis has widespread implications for chemopreventive strategies.

Regulation of breast cancer oncogenesis by the cell of origin's differentiation state.

Human breast cancer which affects 1/8 women is rare at a cellular level. Even in the setting of germline BRCA1/BRCA2, which is present in all breast cells, solitary cancers or cancers arising at only several foci occur. The overwhelming majority of breast cells (109-1012 cells) resist transformation. Our hypothesis to explain this rareness of transformation is that mammary oncogenesis is regulated by the cell of origin's critical window of differentiation so that target cells outside of this window cannot transform. Our novel hypothesis differs from both the multi-hit theory of carcinogenesis and the stem/progenitor cell compartmental theory of tumorigenesis and utilizes two well established murine transgenic models of breast oncogenesis, the FVB/N-Tg (MMTV-PyVT)634Mul/J and the FVB-Tg (MMTV-ErbB2) NK1Mul/J. Tail vein fibroblasts from each of these transgenics were used to generate iPSCs. When select clones were injected into cleared mammary fat pads, but not into non-orthotopic sites of background mice, they exhibited mammary ontogenesis and oncogenesis with the expression of their respective transgenes. iPSC clones, when differentiated along different non-mammary lineages in vitro, were also not able to exhibit either mammary ontogenesis or oncogenesis in vivo. Therefore, in vitro and in vivo regulation of differentiation is an important determinant of breast cancer oncogenesis.

Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes.

Extracellular membrane vesicles (EVs) function as vehicles of intercellular communication, but how the biomaterials they carry reach the target site in recipient cells is an open question. We report that subdomains of Rab7+ late endosomes and nuclear envelope invaginations come together to create a sub-nuclear compartment, where biomaterials associated with CD9+ EVs are delivered. EV-derived biomaterials were also found in the nuclei of host cells. The inhibition of nuclear import and export pathways abrogated the nuclear localization of EV-derived biomaterials or led to their accumulation therein, respectively, suggesting that their translocation is dependent on nuclear pores. Nuclear envelope invagination-associated late endosomes were observed in ex vivo biopsies in both breast carcinoma and associated stromal cells. The transcriptome of stromal cells exposed to cancer cell-derived CD9+ EVs revealed that the regulation of eleven genes, notably those involved in inflammation, relies on the nuclear translocation of EV-derived biomaterials. Our findings uncover a new cellular pathway used by EVs to reach nuclear compartment.

Analogies Between Cancer-Derived Extracellular Vesicles and Enveloped Viruses with an Emphasis on Human Breast Cancer.

PURPOSE OF REVIEW: Cancer cells utilize extracellular vesicles (EVs) as a means of transferring oncogenic proteins and nucleic acids to other cells to enhance the growth and spread of the tumor. There is an unexpected amount of similarities between these small, membrane-bound particles and enveloped virions, including protein content, physical characteristics (i.e., size and morphology), and mechanisms of entry and exit into target cells. RECENT FINDINGS: This review describes the attributes shared by both cancer-derived EVs, with an emphasis on breast cancer-derived EVs, and enveloped viral particles and discusses the methods by which virions can utilize the EV pathway as a means of transferring viral material and oncogenes to host cells. Additionally, the possible links between human papilloma virus and its influence on the miRNA content of breast cancer-derived EVs are examined. SUMMARY: The rapidly growing field of EVs is allowing investigators from different disciplines to enter uncharted territory. The study of the emerging similarities between cancer-derived EVs and enveloped virions may lead to novel important scientific discoveries.

Breast Cancer-Derived Extracellular Vesicles: Characterization and Contribution to the Metastatic Phenotype.

The study of extracellular vesicles (EVs) in cancer progression is a complex and rapidly evolving field. Whole categories of cellular interactions in cancer which were originally presumed to be due solely to soluble secreted molecules have now evolved to include membrane-enclosed extracellular vesicles (EVs), which include both exosomes and shed microvesicles (MVs), and can contain many of the same molecules as those secreted in soluble form but many different molecules as well. EVs released by cancer cells can transfer mRNA, miRNA, and proteins to different recipient cells within the tumor microenvironment, in both an autocrine and paracrine manner, causing a significant impact on signaling pathways, mRNA transcription, and protein expression. The transfer of EVs to target cells, in turn, supports cancer growth, immunosuppression, and metastasis formation. This review focuses exclusively on breast cancer EVs with an emphasis on breast cancer-derived exosomes, keeping in mind that breast cancer-derived EVs share some common physical properties with EVs of other cancers.

A bispecific EpCAM/CD133-targeted toxin is effective against carcinoma.

The discovery of chemoresistant cancer stem cells (CSCs) in carcinomas has created the need for therapies that specifically target these subpopulations of cells. Here, we characterized a bispecific targeted toxin that is composed of two antibody fragments and a catalytic protein toxin allowing it to bind two CSC markers on the same cell killing this resistant subpopulation. CD133 is a well-known CSC marker and has been successfully targeted and caused regression of head and neck squamous cell carcinoma (HNSCC) in vivo. To enable it to bind a broader range of CSCs, an anti-epithelial cell adhesion molecule (EpCAM) scFv was added to create dEpCAMCD133KDEL, a deimmunized bispecific targeted toxin on a single amino acid chain. This bispecific potently inhibited protein translation and proliferation in vitro in three different types of carcinoma. Furthermore, in a CSC spheroid model dEpCAMCD133KDEL eliminated Mary-X spheroids, an inflammatory breast carcinoma. Finally, this bispecific also caused tumor regression in an in vivo model of HNSCC. This represents the first bispecific CSC-targeted toxin and warrants further development as a possible therapy for carcinoma.

Presence of anaplastic lymphoma kinase in inflammatory breast cancer.

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKß in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 µM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK(+) IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics.

The paradox of E-cadherin: role in response to hypoxia in the tumor microenvironment and regulation of energy metabolism.

E-Cadherin is a cell:cell adhesion molecule critical for appropriate embryonic and mammary development. In cancer, E-Cadherin has been primarily viewed as being lost during the process of epithelial-mesenchymal transition (EMT), which occurs with a switch from E-Cadherin expression to a gain of N-Cadherin and other mesenchymal markers. EMT has been shown to play a role in the metastatic process while the reverse process, mesenchymal-epithelial transition (MET), is important for metastatic colonization. Here we report an unexpected role of E-Cadherin in regulating tumorigenicity and hypoxia responses of breast tumors in vivo. Reduced expression of E-Cadherin led to a dramatic reduction of the in vivo growth capability of SUM149, Mary-X and 4T1 tumor cells. Furthermore, over-expression of ZEB1, a known transcriptional repressor of E-Cadherin, led to reduced in vivo growth of SUM149 tumors. Gene set enrichment analysis identified the loss of hypoxia response genes as a major mechanism in mediating the lack of in vivo growth of SUM149 cells that lacked E-Cadherin or over-expressed ZEB1. The in vivo growth defect of SUM149 E-Cadherin knockdown tumors was rescued by the hypoxia-inducible 1α transcription factor (HIF-1α). Given the importance of HIF-1α in cellular metabolism, we observed reduced glycolytic capacity in SUM149 and 4T1 cells that had E-Cadherin knocked down. Our observations shed light on the complex functions of E-Cadherin in retention of an epithelial phenotype and as a mediator of survival of aggressive breast cancer under hypoxic conditions in vivo. Furthermore, we find that patients with basal subtype breast cancer and high E-Cadherin expression in their tumors had a poor clinical outcome. Our data suggests a novel function for E-Cadherin as a bona fide signaling molecule required for the in vivo growth of aggressive breast cancer tumor cells, that retain E-Cadherin expression, in mediating their metabolic function.

The class I HDAC inhibitor Romidepsin targets inflammatory breast cancer tumor emboli and synergizes with paclitaxel to inhibit metastasis.

Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. IBC has distinctive characteristics including invasion of tumor emboli into the skin and rapid disease progression. Given our previous studies suggesting that HDAC inhibitors have promise in targeting IBC, the present study revealed that the class I HDAC inhibitor Romidepsin (FK-288, Istodax; Celgene Corporation, Summit, NJ) potently induced destruction of IBC tumor emboli and lymphatic vascular architecture. associated with inhibition of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha, (HIF1alpha) proteins in the Mary-X pre-clinical model of IBC. Romidepsin treatment induced clinically relevant biomarkers in including induction of acetylated Histone 3 (Ac-H3) proteins, apoptosis, and increased p21WAF1/CIP1. Romidepsin, alone and synergistically when combined with Paclitaxel, effectively eliminated both primary tumors and metastatic lesions at multiple sites formed by the SUM149 IBC cell line. This is the first report of the ability of an HDAC inhibitor to eradicate IBC tumor emboli, to destroy the integrity of lymphatic vessel architecture and to target metastasis. Furthermore, Romidepsin, in combination with a taxane, warrants evaluation as a therapeutic strategy that may effectively target the skin involvement and rapid metastasis that are hallmarks of IBC.

Early to intermediate steps of tumor embolic formation involve specific proteolytic processing of E-cadherin regulated by Rab7.

The lymphovascular embolus is an enigmatic entity adept at metastatic dissemination and chemotherapy resistance. Using MARY-X, a human breast cancer xenograft that exhibits florid lymphovascular emboli in mice and spheroids in vitro, we established a model where the in vitro transition stages from minced tumoral aggregates to well-formed spheroids served as a surrogate for in vivo emboli formation. MARY-X well-formed spheroids and emboli exhibited strong similarity of expression. The aggregate-to-spheroid transition stages were characterized by increased ExoC5, decreased Hgs and Rab7, increased calpains, increased full-length E-cadherin (E-cad/FL), and the transient appearance of E-cad/NTF2, a 95 kDa E-cadherin fragment and increased Notch3icd (N3icd), the latter two fragments produced by increased γ-secretase. Both transient and permanent knockdowns of Rab7 in MCF-7 cells increased protein but not transcription of E-cad/FL and resulted in the de novo appearance of E-cad/NTF2, the presence of nuclear E-cad/CTF2, and increased Notch1icd (N1icd). Overexpression of Rab7 conversely decreased E-cad/FL, γ-secretase (PS1/NTF), and E-cad/NTF2. Overexpression of calpains did not alter PS1/NTF but decreased E-cad/FL and E-cad/NTF2 and increased N1icd. Well-formed spheroids showed increased Rab7, absent E-cad/NTF2, decreased PS1/NTF, increased E-cad/NTF1, and increased N3icd, the latter two fragments being the direct and indirect consequences, respectively, of increased calpains (calpain 1 and calpain 2). Inhibition of calpains decreased E-cad/NTF1 but increased E-cad/NTF2 showing that calpains compete with γ-secretase (PS1) for closely located cleavage/binding sites on E-cadherin and that increased calpains can shuttle even decreased levels of γ-secretase to Notch 3, resulting in increased Notch 3 signaling in the well-formed spheroids.

Singular v dual inhibition of SNF2L and its isoform, SNF2LT, have similar effects on DNA damage but opposite effects on the DNA damage response, cancer cell growth arrest and apoptosis.

SNF2L, an ATPase chromatin remodeling gene nearly ubiquitously expressed in diverse tissues, cancers, and derived cell lines, contributes to the chromatin remodeling complex that facilitates transcription. Because of this near ubiquitous expression, it has not been exploited as a cancer therapeutic target. However, in a recent study, we found that highly malignant cancer cells, although expressing SNF2L at similar levels as their normal counterparts, were sensitive to its knockdown. Only the highly malignant (HM) lines showed significant growth inhibition, DNA damage, a DNA damage response, and phosphorylation of checkpoint proteins and marked apoptosis. In studying SNF2L, we discovered a novel truncated isoform, SNF2LT which, when compared to full length SNF2L, lacked three important domains: HAND, SANT and SLIDE. Although truncated isoforms usually have antagonistic functions to their parental molecule, here SNF2LT knockdown had similar effects to the knockdown of its parental molecule, SNF2L, of inducing DNA damage, a DNA damage response, cell cycle arrest and apoptosis selectively in cancer cell lines. However dual SNF2L and SNF2LT knockdown, while inducing DNA damage, did not result in a DNA damage response, a cell cycle arrest and apoptosis. In fact HM lines subjected to dual knockdown paradoxically exhibited sustained cell growth. Our findings indicate that the ratio of SNF2L to its isoform tightly regulates the cancer cell's response to DNA damage. Cancer cell lines which endogenously express low levels of both SNF2L and its isoform mimic the situation of dual knockdown and permit DNA damage which is allowed to propagate unchecked.

KIAA0101 interacts with BRCA1 and regulates centrosome number.

To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control.

E-cadherin accumulation within the lymphovascular embolus of inflammatory breast cancer is due to altered trafficking.

E-Cadherin functions as a tumor suppressor in some invasive breast carcinomas and metastasis is promoted when its expression is lost. It has been observed, however, that in one of the most aggressive human breast cancers, inflammatory breast cancer (IBC), E-cadherin is overexpressed and this accounts for the formation of the lymphovascular embolus, a structure efficient at metastasis and resistant to chemotherapy through unknown cytoprotective mechanisms. Studies using a human xenograft model of IBC, MARY-X, indicate that the mechanism of E-cadherin overexpression is not transcriptional but related to altered protein trafficking. By real-time RT-PCR, E-cadherin transcript levels in MARY-X were 3- to 11-fold less than in other E-cadherin positive human breast carcinoma lines but the protein levels were 5- to 10-fold greater. In addition, several smaller E-cadherin protein fragments, e.g. 95 kDa, were present. To explain these observations, it was hypothesized that there may be altered protein trafficking. A real-time RT-PCR screen of candidate molecules generally known to regulate protein trafficking was conducted. The screen revealed 3.5- to 7-fold increased ExoC5 level and 10 to 20 fold decreased HRS and RAB7 levels, which was confirmed in human microdissected lymphovascular emboli. Since these alterations may only be correlative with E-cadherin overexpression, one of the molecules, Rab7, was selectively knocked down in MCF-7 cells. An increase in the full length 120 kDa E-cadherin and the de novo appearance of the 95 KD band were observed. These findings suggest that it is the altered E-cadherin trafficking that contributes to its oncogenic rather than suppressive role in IBC.