BACKGROUND:While neoadjuvant endocrine therapy (NET), with or without a CDK4/6 inhibitor, is an established treatment option for estrogen receptor-positive breast cancers, optimal patient selection and second-line treatment for non-responders remain uncertain. METHODS:In the open-label, phase 2 PETREMAC trial (NCT02624973), pre- and postmenopausal patients with large T2 (>4 cm) or locally advanced ER/PGR>50 %, HER2-, and TP53 wild-type breast cancers received NET (tamoxifen + goserelin for premenopausal and letrozole for postmenopausal patients). Palbociclib was added if the Ki67 reduction was ≤50 % after 14 days. Neoadjuvant chemotherapy (NAC) was introduced if NET ± palbociclib failed to reduce Ki67 sufficiently or if there was no objective response on MRI after 24 weeks. Tumor biopsies underwent targeted sequencing of 360 cancer-related genes and subsequent gene expression profiling. RESULTS:Among 88 patients, the median tumor size was 48 mm (range 16-140 mm). NET alone reduced Ki67 > 50 % in 49/88 (56 %) of evaluable tumors. Adding palbociclib yielded a Ki67 reduction >50 % in 24/34 (71 %) of tumors where neoadjuvant endocrine therapy alone failed to suppress Ki67, providing a Ki67 reduction of >50 % in a total of 72/88 (82 %) of patients. NAC was administered to 34/88 (39 %) due to inadequate Ki67 response or lack of MRI response. Overall, 70 % achieved a pre-surgical objective response. Pathological complete response was seen in 3/84 patients. Postmenopausal status (p = 0.005) and invasive lobular carcinoma (p = 0.02) predicted Ki67-based response to NET. CONCLUSION:Sequential NET with palbociclib, limiting NAC to non-responders, is a feasible strategy for ER/PGR>50 %, HER2-, TP53 wild-type breast cancers.

Abstract Background: Low level normal cell BRCA1 epimutations have been associated with an increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance pattern of BRCA1 epimutations established. Methods: To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 411 patients with primary breast cancer, including 66 TNBCs. Samples were analyzed by a highly sensitive next-generation sequencing (NGS) assay on an Illumina MiSeq sequencer, allowing allele-resolved methylation assessment. Further, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord samples from 1260 newborn girls and 200 newborn boys.To assess potential Mendelian heritage, we analyzed BRCA1 methylation status in WBCs from 575 mothers and 531 fathers of newborn girls with (n = 102) and without (n = 473) WBC BRCA1 methylation. Results: We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (< 10%) of tumors (combined 14 out of 72; 19.4%, CI: 11.1-30.5). These exceeded the number of tumors harboring germline (n = 5) or somatic (n = 4) BRCA1 mutations. Notably, BRCA1 methylation and BRCA1 mutations were mutually exclusive. Contrasting the findings in TNBC and ER-low exprssion tumors, we found WBC and tumor BRCA1 methylation concordance in only three out of 221 patients with ER >10+% tumors and zero out of 116 patients with HER2 positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical cord WBCs from newborn girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals. We found no correlation between WBC BRCA1 methylation in newborns and methylation status in their mothers, fathers, or any parent. Notably, WBC BRCA1 methylation occurred at a significantly lower frequency in newborn boys ( 9/200; 4.5%) as compared to newborn girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46/575; 8.0%; p = 0.0003). Conclusions: Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expressing breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance. Citation Format: Per Lonning, Hans Petter Eikesdal, Elisabet Ognedal, Bjornar Gilje, Steinar Lundgren, Egil Blix, Helge Espelid, Jürgen Geisler, Stephanie Geisler, Emiel Janssen, Synnøve Yndestad, Laura Minsaas, Beryl Leirvaag, Reidun Lillestol, Stian Knappskog, Oleksii Nikolaienko. Prenatal BRCA1 epimutations is a major cause of triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-09.

PURPOSE:Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. METHODS:HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials.gov identifier: NCT02624973). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. RESULTS:HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P < .001). CONCLUSION:The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD.

Abstract Background Normal cell BRCA1 epimutations have been associated with increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance patterns of BRCA1 epimutations established. Methods To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 408 patients with 411 primary breast cancers, including 66 TNBCs, applying a highly sensitive sequencing assay, allowing allele-resolved methylation assessment. Furthermore, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord blood of 1260 newborn girls and 200 newborn boys. Finally, we assessed BRCA1 methylation status among 575 mothers and 531 fathers of girls with (n = 102) and without (n = 473) BRCA1 methylation. Results We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (< 10%) tumors (combined: 14 out of 72; 19.4%; 95% CI 11.1–30.5). In contrast, we found concordant WBC and tumor methylation in only three out of 220 patients with 221 ER ≥ 10% tumors and zero out of 114 patients with 116 HER2-positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical WBCs from girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals, but no correlation to BRCA1 methylation status either in mothers or fathers. A significantly lower fraction of newborn boys carried BRCA1 methylation (9/200; 4.5%) as compared to girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46/575; 8.0%; p = 0.0003). Conclusions Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expression breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance.

Abstract Background: Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and predictive of response to PARP inhibition in the primary setting (Eikesdal et al, Ann Oncol, 2021). However, the prevalence of HRD across breast cancer subtypes has not been established. Methods: Pretreatment tumor biopsies from 201 patients (32 TNBC and 169 non-TNBC) with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials #NCT02624973) were examined. These samples underwent targeted cancer gene panel sequencing and BRCA1 promoter methylation analysis to assess HRD status defined by homologous recombination repair (HRR) gene mutations and/or BRCA1 promoter methylation. HRR genes included BRCA1, BRCA2, BRIP1, BARD1, and PALB2 by strict definition (HRR-S), and additionally ABL1, ATM, ATR, ATRX, BLM, CDK12, CHEK1, EMSY, ERCC4, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, MEN1, MRE11, NBN, PTEN, and SETD2 by wider definition (HRR-W). HRD strict (HRD-S) was defined as biallelic gene inactivation by HRR-S mutations or BRCA1 methylation. Finally, tumors underwent PAM50 gene expression subtyping and evaluation of functional HRD by RAD51 nuclear foci analysis, for which a low score has been associated with HRD. Results: HRD-S was present in 13% of the breast cancers (total: n= 27/201; TNBC: 15/32; 47%; non-TNBC: 12/169; 7%), whereas HRD-W (HRR-W or BRCA1 methylation) was observed in 29% (total: n=58/201; TNBC: 19/32; 59%; non-TNBC: 39/169; 23%). Among 190 tumors analyzed for PAM50 intrinsic subtype, HRD-S was detected in 3/60 and 4/48 (5% and 8%) of tumors classified as luminal A and B, respectively, 1/35 (3%) of HER2-enriched, 4/21 (19%) of normal-like, and 12/26 (46%) of basal-like tumors. Out of 58 non-TNBC biopsies examined by RAD51 staining, four (7%) were classified as HRD-S and all these were scored as RAD51 low. The remaining 54 non-TNBC samples were homologous recombination proficient, and none of these exhibited functional HRD by RAD51 low scores. All four HRD-S/RAD51 low tumors were hormone receptor-positive, HER2 negative, and belonged to the luminal A (n=1), luminal B (n=2), and basal-like (n=1) subtypes, with HRD caused by germline BRCA1 (gBRCA1), gBRCA2, somatic BRCA1 mutations and BRCA1 methylation, respectively. Conclusion: The prevalence of HRD across all breast cancer subtypes suggests that HRD analysis and therapy targeting such DNA repair defects should be tested in future clinical trials. Citation Format: Christina Engebrethsen, Synnøve Yndestad, Andrea Herencia-Ropero, Oleksii Nikolaienko, Olav Karsten Vintermyr, Reidun K. Lillestøl, Laura Minsaas, Beryl Leirvaag, Gjertrud Iversen, Bjørnar Gilje, Egil Blix, Helge Espelid, Steinar Lundgren, Jürgen Geisler, Liv Jorunn Vassbotn, Hildegunn S. Aase, Turid Aas, Alba Llop-Guevara, Violeta Serra, Per Eystein Lønning, Stian Knappskog, Hans Petter Eikesdal. Homologous recombination deficiency across subtypes of primary breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-04.

Background: The antitumor efficacy of PARP inhibitors (PARPi) for breast cancer patients harboring germline BRCA1/2 (gBRCA1/2) mutations is well established. While PARPi monotherapy was ineffective in patients with metastatic triple negative breast cancer (TNBC) wild type for BRCA1/2, we hypothesized that PARPi may be effective in primary TNBCs without previous chemotherapy exposure. Patients and methods: In the phase II PETREMAC trial, patients with primary TNBC >2 cm received olaparib for up to 10 weeks before chemotherapy. Tumor biopsies collected before and after olaparib underwent targeted DNA sequencing (360 genes) and BRCA1 methylation analyses. In addition, BRCAness (multiplex ligation-dependent probe amplification), PAM50 gene expression, RAD51 foci, tumor-infiltrating lymphocytes (TILs) and PD-L1 analyses were performed on pretreatment samples. Results: The median pretreatment tumor diameter was 60 mm (range 25-112 mm). Eighteen out of 32 patients obtained an objective response (OR) to olaparib (56.3%). Somatic or germline mutations affecting homologous recombination (HR) were observed in 10/18 responders [OR 55.6%, 95% confidence interval (CI) 33.7-75.4] contrasting 1/14 non-responders (OR 7.1%; CI 1.3-31.5, P = 0.008). Among tumors without HR mutations, 6/8 responders versus 3/13 non-responders revealed BRCA1 hypermethylation (P = 0.03). Thus, 16/18 responders (88.9%, CI 67.2-96.9), in contrast to 4/14 non-responders (28.6%, CI 11.7-54.7, P = 0.0008), carried HR mutations and/or BRCA1 methylation. Excluding one gPALB2 and four gBRCA1/2 mutation carriers, 12/14 responders (85.7%, CI 60.1-96.0) versus 3/13 non-responders (23.1%, CI 8.2-50.3, P = 0.002) carried somatic HR mutations and/or BRCA1 methylation. In contrast to BRCAness signature or basal-like subtype, low RAD51 scores, high TIL or high PDL1 expression all correlated to olaparib response. Conclusion: Olaparib yielded a high clinical response rate in treatment-naive TNBCs revealing HR deficiency, beyond germline HR mutations.

An increasing number of researchers consider changes in DNA methylation at the promoter regions of specific loci to be as important as genetic events in the initiation of cancer (1). The seminal publication that implicated promoter region methylation (hereafter referred to as methylation) as an initiating event in carcinogenesis was the report that methylation of a familial cancer driver, the RB1 tumor suppressor gene, was observed in nonfamilial retinoblastomas (2). Inactivation of the gene by epigenetic silencing, leading to functional loss of activity, thus was an alternative to mutational inactivation. It then became apparent that other genes that recurrently mutated in certain types of familial cancer, such as BRCA1, MLH1, and CDKN2A, could be methylated in the corresponding sporadic cancers. This finding indicated that inactivation of these genes by methylation might be the critical driver event in the development of those cases. Originally, BRCA1 was thought to play only a small role in nonfamilial cancer, because coding region mutations in sporadic breast and ovarian cancer occur very infrequently. Subsequently, the BRCA1 promoter region was found to be methylated in a proportion of sporadic cases of both breast and ovarian cancer (3, 4). Of note, BRCA1 methylation is rare outside these 2 tumor types, indicating that BRCA1 methylation may indeed play an important role in pathogenesis (3, 4). Another paradigm shift occurred with the observation in a series of microsatellite-unstable colorectal cancer that allelic MLH1 methylation in peripheral white blood cells (WBCs) might be seen in some patients with tumor MLH1 methylation (5). Loss of heterozygosity of the unmethylated MLH1 allele in the tumor implicated the methylated MLH1 allele as the tumorigenic driver. Subsequently, BRCA1 promoter methylation was shown to be present in the WBCs of some patients who had breast cancer in which the tumors had a morphologic characteristic the same as that in patients with BRCA1 mutation (6), again implicating the methylation event as the tumorigenic driver. These findings suggested a model for predisposition to cancer in which a soma-wide or constitutional distribution of an epigenetic driver event acts as the first hit in tumorigenesis. The term constitutional methylation (not to be confused with constitutive methylation) refers to cases in which aberrant methylation of a given region occurs in one or more tissues in the body outside the tumor or peritumoral region. Of interest, while MLH1 methylation usually is allelic (50%), BRCA1 methylation in peripheral WBCs typically is mosaic, with overall methylated allele frequencies ranging from 15% to less than 1%. A key study used a population-based cohort of women with early-onset breast cancer whose tumors had been scored for pathologic features associated with BRCA1-mutant tumors (7). Constitutional methylation was found in the WBCs of more than 30% of the women with BRCA1-like breast cancer, whereas it was present in only a small percentage (3% or 4%) of those with nonBRCA1-like cancer and in control participants. White blood cell BRCA1 methylation was associated with an increased risk for early-onset breast cancer. The cancer cases that arose were methylated for BRCA1 at elevated levels consistent with expansion of a clonal BRCA1 methylation event (7). Because BRCA1 mutation drives both triple-negative breast and high-grade serous ovarian cancer, it is reasonable to consider that similar mechanisms would be in play in the latter. In their current Annals article, Lnning and colleagues (8) report the presence of detectable BRCA1 methylation in the WBCs of patients with ovarian cancer compared with control participants, as determined in 2 independent casecontrol studies derived from the Norwegian population. This investigation is admirable not only because of the large number of patients and control participants studied, but also because the investigators conducted additional research to interpret their findings. In particular, they showed that tumor burden is not a contributor to the levels of BRCA1 methylation in WBCs, confirming that methylation is intrinsic to these cells rather than the result of contaminating tumor cells. BRCA1 methylation also was shown in other, non-WBC tissues, unrelated to the tumor, further supporting the concept of constitutional methylation. The Lnning group (8) observed that about 4% of unaffected control participants had detectable BRCA1 methylation in their WBCs. This contrasts with the finding of WBC methylation in more than 9% of patients with high-grade serous ovarian cancer, whereas patients with other ovarian cancer subtypes had WBC methylation frequencies similar to those of the unaffected control participants (8). This mirrors the previous observations in patients with breast cancer that elevated WBC BRCA1 methylation frequency was restricted to those with BRCA1-like cancer (7). An interesting finding in the current study involved changes in BRCA1 methylation frequency with aging. The frequency in newborns was 7% but declined continually over time, with decreases occurring in both patients and control populations. Tumor tissue was available for 87 patients in the present study. Tumor methylation was observed in 19 of 29 patients with WBC BRCA1 methylation but only 7 of 58 without it. These findings are consistent with those of a similar, smaller investigation using patients from the Australian Ovarian Cancer Study (9). Although the current study did not report tumor methylation levels (8), one would expect most of the tumors arising in patients with WBC BRCA1 methylation to show high levels of methylation. This and previous studies make it clear that BRCA1 constitutional methylation is strongly associated with the same tumor types as those with mutation of the gene. BRCA1 constitutional methylation is an alternate mechanism of BRCA1 inactivation that leads to an increased risk for breast or ovarian cancer that is methylated for BRCA1. Although it is clear that WBC BRCA1 methylation is associated with an elevated risk for breast or ovarian cancer, the disease will develop in only some of the otherwise healthy women with constitutional methylation. Is this risk stochastically or environmentally driven? What is the cause of constitutional BRCA1 methylation? The answers to these questions may have important implications for predicting and preventing these types of cancer in the future (10).

Background: Filamin A (FLNa) is an actin-crosslinking protein necessary for stabilizing the cell surface, organizing protrusive activity and for promoting efficient cellular translocation. Recently, our group demonstrated the requirement of FLNa for the internalization of the chemokine receptor CCR2B.Methodology and Principal Findings: In order to study the role of FLNa in vitro and in real-time, we have developed a fluorescent FLNa-EGFP construct. In this novel imaging tool, we introduced the EGFP-tag inside the flexible hinge 1 region of FLNa between two calpain cleavage sites. Our findings indicate that the FLNa-EGFP construct was correctly expressed, cleaved by calpain and colocalized with actin filaments as shown by immunostaining experiments in the human melanoma cell lines A7 (FLNa-repleted) and M2 (FLNa-deficient). In addition, scanning-electron microscopy (SEM) and micropatterning studies also provided clear evidence that the cell rigidity was restored. FLNa-EGFP allowed us to demonstrate the interaction of FLNa with the chemokine receptor CCR2B in endocytic vesicles after CCL2 ligand stimulation. Through live-cell imaging studies we show that the CCR2B receptor in Rab5-positive vesicles moves along filamin A-positive fibers.Significance: Taken together, these results outline the functionality of the FLNa-EGFP and the importance of filamin A for receptor internalization and movement into endocytic vesicles.

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.

The technology of UniTargetingResearch AS (UTRtech (TM)) is based on the finding that the efficiency of directing mRNA to the endoplasmic reticulum is influenced by targeting signals. Using selected signals, genetically engineered mammalian cells are generated from which a protein of interest can be efficiently secreted. An industrial collaboration has revealed that UTRtech (TM) has the potential to significantly enhance the production of therapeutic proteins.

Metabolically unstable proteins are involved in a multitude of regulatory networks, including those that control cell signaling, the cell cycle and in many responses to physiological stress. In the present study, we have determined the stability and characterized the degradation process of some members of the G(q) class of heterotrimeric G proteins. Pulse-chase experiments in HEK293 cells indicated a rapid turnover of endogenously expressed G alpha(q) and overexpressed G alpha(q) and G alpha(16) subunits. Pretreatment with proteasome inhibitors attenuated the degradation of both G alpha subunits. In contrast, pretreatment of cells with inhibitors of lysosomal proteases and nonproteasomal cysteine proteases had very little effect on the stability of the proteins. Significantly, the turnover of these proteins is not affected by transient activation of their associated receptors. Fractionation studies showed that the rates of G alpha(q) and G alpha(16) degradation are accelerated in the cytosol. In fact, we show that a mutant G alpha(q) which lacks its palmitoyl modification site, and which is localized almost entirely in the cytoplasm, has a marked increase in the rate of degradation. Taken together, these results suggest that the G(q) class proteins are degraded through the proteasome pathway and that cellular localization and/or other protein interactions determine their stability.

Metabolically unstable proteins are involved in a multitude of regulatory networks, including those that control cell signaling, the cell cycle and in many responses to physiological stress. In the present study, we have determined the stability and characterized the degradation process of some members of the Gq class of heterotrimeric G proteins. Pulse‐chase experiments in HEK293 cells indicated a rapid turnover of endogenously expressed Gαq and overexpressed Gαq and Gα16 subunits. Pretreatment with proteasome inhibitors attenuated the degradation of both G alpha subunits. In contrast, pretreatment of cells with inhibitors of lysosomal proteases and nonproteasomal cysteine proteases had very little effect on the stability of the proteins. Significantly, the turnover of these proteins is not affected by transient activation of their associated receptors. Fractionation studies showed that the rates of Gαq and Gα16 degradation are accelerated in the cytosol. In fact, we show that a mutant Gαq which lacks its palmitoyl modification site, and which is localized almost entirely in the cytoplasm, has a marked increase in the rate of degradation. Taken together, these results suggest that the Gq class proteins are degraded through the proteasome pathway and that cellular localization and/or other protein interactions determine their stability.